Enzyme treatment of foodstuffs for celiac sprue

ABSTRACT

Administering an effective dose of glutenase to a Celiac or dermatitis herpetiformis patient reduces levels of toxic gluten oligopeptides, thereby attenuating or eliminating the damaging effects of gluten.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional application60/357,238 filed Feb. 14, 2002; to U.S. Provisional Application60/380,761 filed May 14, 2002; to U.S. Provisional Application60/392,782 filed Jun. 28, 2002; and to U.S. Provisional application No.60/422,933, filed Oct. 31, 2002, to U.S. Provisional Application60/428,033, filed Nov. 20, 2002, and to U.S. Provisional Application60/435,881, filed Dec. 20, 2002, each of which are herein specificallyincorporated by reference.

BACKGROUND OF THE INVENTION

[0002] In 1953, it was first recognized that ingestion of gluten, acommon dietary protein present in wheat, barley and rye causes diseasein sensitive individuals. Gluten is a complex mixture of glutamine- andproline-rich glutenin and prolamine molecules, which is thought to beresponsible for disease induction. Ingestion of such proteins bysensitive individuals produces flattening of the normally luxurious,rug-like, epithelial lining of the small intestine known to beresponsible for efficient and extensive terminal digestion of peptidesand other nutrients. Clinical symptoms of Celiac Sprue include fatigue,chronic diarrhea, malabsorption of nutrients, weight loss, abdominaldistension, anemia, as well as a substantially enhanced risk for thedevelopment of osteoporosis and intestinal malignancies (lymphoma andcarcinoma). The disease has an incidence of approximately 1 in 200 inEuropean populations.

[0003] A related disease is dermatitis herpetiformis, which is a chroniceruption characterized by clusters of intensely pruritic vesicles,papules, and urticaria-like lesions. IgA deposits occur in almost allnormal-appearing and perilesional skin. Asymptomatic gluten-sensitiveenteropathy is found in 75 to 90% of patients and in some of theirrelatives. Onset is usually gradual. Itching and burning are severe, andscratching often obscures the primary lesions with eczematization ofnearby skin, leading to an erroneous diagnosis of eczema. Strictadherence to a gluten-free diet for prolonged periods may control thedisease in some patients, obviating or reducing the requirement for drugtherapy. Dapsone, sulfapyridine and colchicines are sometimes prescribedfor relief of itching.

[0004] Celiac Sprue is generally considered to be an autoimmune diseaseand the antibodies found in the serum of the patients supports a theoryof an immunological nature of the disease. Antibodies to tissuetransglutaminase (tTG) and gliadin appear in almost 100% of the patientswith active Celiac Sprue, and the presence of such antibodies,particularly of the IgA class, has been used in diagnosis of thedisease.

[0005] The large majority of patients express the HLA-DQ2 [DQ(a1*0501,b1*02)] and/or DQ8 [DQ(a1*0301, b1*0302)] molecules. It is believed thatintestinal damage is caused by interactions between specific gliadinoligopeptides and the HLA-DQ2 or DQ8 antigen, which in turn induceproliferation of T lymphocytes in the sub-epithelial layers. T helper 1cells and cytokines apparently play a major role in a local inflammatoryprocess leading to villus atrophy of the small intestine.

[0006] At the present time there is no good therapy for the disease,except to completely avoid all foods containing gluten. Although glutenwithdrawal has transformed the prognosis for children and substantiallyimproved it for adults, some people still die of the disease, mainlyadults who had severe disease at the outset. An important cause of deathis lymphoreticular disease (especially intestinal lymphoma). It is notknown whether a gluten-free diet diminishes this risk. Apparent clinicalremission is often associated with histologic relapse that is detectedonly by review biopsies or by increased EMA titers.

[0007] Gluten is so widely used, for example in commercial soups,sauces, ice creams, hot dogs, and other foods, that patients needdetailed lists of foodstuffs to avoid and expert advice from a dietitianfamiliar with celiac disease. Ingesting even small amounts of gluten mayprevent remission or induce relapse. Supplementary vitamins, minerals,and hematinics may also be required, depending on deficiency. A fewpatients respond poorly or not at all to gluten withdrawal, eitherbecause the diagnosis is incorrect or because the disease is refractory.In the latter case, oral corticosteroids (e.g., prednisone 10 to 20 mgbid) may induce response.

[0008] In view of the serious and widespread nature of Celiac Sprue,improved methods of treating or ameliorating the effects of the diseaseare needed. The present invention addresses such needs.

SUMMARY OF THE INVENTION

[0009] The present invention provides methods for treating the symptomsof Celiac Sprue and/or dermatitis herpetiformis by decreasing the levelsof toxic gluten oligopeptides in foodstuffs, either prior to or afteringestion by a patient. The present invention relates to the discoverythat certain gluten oligopeptides resistant to cleavage by gastric andpancreatic enzymes, that the presence of such peptides results in toxiceffects, and that enzymatic treatment can remove such peptides and theirtoxic effects. By digestion with glutenases, these toxic oligopeptidesare cleaved into fragments, thereby preventing or relieving their toxiceffects in Celiac Sprue or dermatitis herpetiformis patients. In oneaspect of the invention, a foodstuff is treated with a glutenase priorto consumption by the patient. In another aspect of the invention, aglutenase is administered to a patient and acts internally to destroythe toxic oligopeptides. In another aspect of the invention, arecombinant organism that produces a glutenase is administered to apatient. In another aspect of the invention, gene therapy is used toprovide the patient with a gene that expresses a glutenase that destroysthe toxic oligopeptides.

[0010] In one aspect, the invention provides methods for theadministration of enteric formulations of one or more glutenases, eachof which may be present as a single agent or a combination of activeagents. In another aspect of the invention, stabilized forms ofglutenases are administered to the patient, which stabilized forms areresistant to digestion in the stomach, e.g. to acidic conditions.Alternative methods of administration include genetic modification ofpatient cells, e.g. enterocytes, to express increased levels ofpeptidases capable of cleaving immunogenic oligopeptides of gliadin;pretreatment of foods with glutenases; the introduction ofmicro-organisms expressing such peptidases so as to transiently orpermanently colonize the patient intestinal tract; and the like.

[0011] In another aspect, the invention provides pharmaceuticalformulations containing one or more glutenases and a pharmaceuticallyacceptable carrier. Such formulations include formulations in which theglutenase is contained within an enteric coating that allows delivery ofthe active agent to the intestine and formulations in which the activeagents are stabilized to resist digestion in acidic stomach conditions.The formulation may comprise one or more glutenases or a mixture or“cocktail” of agents having different activities.

[0012] In another aspect, the invention provides foodstuffs derived fromgluten-containing foods that have been treated to remove or to reduce tonon-toxic levels the gluten-derived oligopeptides that are toxic toCeliac Sprue patients, and methods for treating foods to hydrolyze toxicgluten oligopeptides. In other aspects, the invention providesrecombinant microorganisms useful in hydrolyzing the gluten-derivedoligopeptides that are toxic to Celiac Sprue patients from foodstuffs;methods for producing glutenases that digest the gluten-derivedoligopeptides that are toxic to Celiac Sprue patents; purifiedpreparations of the glutenases that digest the gluten-derivedoligopeptides that are toxic to Celiac Sprue patents; and recombinantvectors that code for the expression of glutenases that digest thegluten-derived oligopeptides that are toxic to Celiac Sprue patents.

[0013] These and other aspects and embodiments of the invention aredescribed in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS. 1A-1B. Brush border membrane catalyzed digestion of theimmunodominant gliadin peptide. FIG. 1A: LC-MS traces of peptides asshown, after digestion with 27 ng/μl rat brush border membrane (BBM)protein for the indicated time. Reaction products were separated byreversed phase HPLC and detected by mass spectroscopy (ion countsm/z=300-2000 g/mol). The indicated peptide fragments were confirmed bycharacteristic tandem MS fragmentation patterns. The SEQ ID NO:2pyroQLQPFPQPQLPY peak corresponds to an N-terminally pyroglutaminatedspecies, which is generated during HPLC purification of the syntheticstarting material. FIG. 1B: Abundance of individual digestion productsas a function of time. The peptide fragments in FIG. 1A were quantifiedby integrating the corresponding MS peak area (m/z=300-2000 g/mol). Theresulting MS intensities are plotted as a function of digestion time(with BBM only). The digestion experiment was repeated in the presenceof exogenous DPP IV from Aspergillus fumigatus (Chemicon International,CA, 0.28 μU DPP IV/ng BBM protein) and analyzed as above (open bars).The relative abundance of different intermediates could be estimatedfrom the UV₂₈₀ traces and control experiments using authentic standards.The inserted scheme shows an interpretative diagram of the digestionpathways of SEQ ID NO:1) QLQPFPQPQLPY and its intermediates, the BBMpeptidases involved in each step, and the amino acid residues that arereleased. The preferred breakdown pathway is indicated in bold.APN=aminopeptidase N, CPP=carboxypeptidase P, DPP IV=dipeptidyldipeptidase IV.

[0015] FIGS. 2A-2B. C-terminal digestion of the immunodominant gliadinpeptide by brush border membrane. FIG. 2A: (SEQ ID NO:3) PQPQLPYPQPQLPYwas digested by 27 ng/μl brush border membrane (BBM) proteinpreparations for the indicated time and analyzed as in FIG. 1A. Theidentity of the starting material and the product (SEQ ID NO:4)PQPQLPYPQPQLP was corroborated by MSMS fragmentation. The intrinsic massintensities of the two peptides were identical, and the UV₂₈₀ extinctioncoefficient of (SEQ ID NO:4) PQPQLPYPQPQLP was half of the startingmaterial in accordance with the loss of one tyrosine. All otherintermediates were ≦1%. The scheme below shows the proposed BBMdigestion pathway of (SEQ ID NO:3) PQPQLPYPQPQLPY with no observedN-terminal processing (crossed arrow) and the removal of the C-terminaltyrosine by carboxypeptidase P (CPP) in bold. Further C-terminalprocessing by dipeptidyl carboxypeptidase (DCP) was too slow to permitanalysis of the subsequent digestion steps (dotted arrows). FIG. 2B:Influence of dipeptidyl carboxypeptidase on C-terminal digestion. (SEQID NO:3) PQPQLPYPQPQLPY in phosphate buffered saline:Tris bufferedsaline=9:1 was digested by BBM alone or with addition of exogenousrabbit lung DCP (Cortex Biochemicals, CA) or captopril. After overnightincubation, the fraction of accumulated SEQ ID NO:4) PQPQLPYPQPQLP(compared to initial amounts of (SEQ ID NO:3) PQPQLPYPQPQLPY at t=0 min)was analyzed as in FIG. 2A, but with an acetonitrile gradient of 20-65%in 6-35 minutes.

[0016]FIG. 3. Dose dependent acceleration of brush border mediateddigestion by exogenous endoproteases. As seen from FIGS. 2A-2B, thepeptide (SEQ ID NO:4) PQPQLPYPQPQLP is stable toward further digestion.This peptide was digested with 27 ng/μl brush border membranes, eitheralone, with increasing amounts of exogenous prolyl endopeptidase (PEP,specific activity 28 μU/pg) from Flavobacterium meningosepticum (USBiological, MA), or with additional elastase (E-1250, Sigma, MO),bromelain (B-5144, Sigma, MO) or papain (P-5306, Sigma, MO) (12). Afterone hour, the fraction of remaining (SEQ ID NO:4) PQPQLPYPQPQLP(compared to the initial amount at t=0 min) was analyzed and quantifiedas in FIG. 1.

[0017]FIG. 4. Products of gastric and pancreatic protease mediateddigestion of α2-gliadin under physiological conditions. Analysis wasperformed by LC-MS. The longest peptides are highlighted by arrows andalso in the sequence of α2-gliadin (inset).

[0018]FIG. 5. In vivo brush border membrane digestion of peptides.LC-UV₂₁₅ traces of 25 μM of (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF before perfusion and after perfusion(residence time=20 min). LC-UV₂₁₅ traces of 50 μM of SEQ ID NO:1QLQPFPQPQLPY before perfusion and after perfusion (residence time=20min).

[0019]FIG. 6. Alignment of representative gluten and non-gluten peptideshomologous to (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.

[0020]FIG. 7. Breakdown and detoxification of 33-mer gliadin peptidewith PEP. In vitro incubation of PEP (540 mU/ml) with the 33-mer gliadinpeptide (100 μM) for the indicated time. In vivo digestion of the 33-mergliadin peptide (25 μM) with PEP (25 mU/ml) and the rat's intestine(residence time=20 min).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] Celiac Sprue and/or dermatitis herpetiformis are treated bydigestion of gluten oligopeptides contained in foodstuffs consumed byindividuals suffering from one or both conditions. Gluten oligopeptidesare highly resistant to cleavage by gastric and pancreatic peptidasessuch as pepsin, trypsin, chymotrypsin, and the like. By providing fordigestion of gluten oligopeptides with glutenase, oligopeptides arecleaved into fragments, thereby preventing the disease-causing toxicity.

[0022] Methods and compositions are provided for the administration ofone or more glutenases inhibitors to a patient suffering from CeliacSprue and/or dermatitis herpetiformis. In some patients, these methodsand compositions will allow the patient to ingest glutens withoutserious health consequences, much the same as individuals that do notsuffer from either of these conditions. In some embodiments, theformulations of the invention comprise a glutenase contained in anenteric coating that allows delivery of the active agent(s) to theintestine; in other embodiments, the active agent(s) is stabilized toresist digestion in acidic stomach conditions. In some cases the activeagent(s) have hydrolytic activity under acidic pH conditions, and cantherefore initiate the proteolytic process on toxic gluten sequences inthe stomach itself. Alternative methods of administration provided bythe invention include genetic modification of patient cells, e.g.enterocytes, to express increased levels of glutenases; and theintroduction of micro-organisms expressing such glutenases so as totransiently or permanently colonize the patient's intestinal tract. Suchmodified patient cells (which include cells that are not derived fromthe patient but that are not immunologically rejected when administeredto the patient) and microorganisms of the invention are, in someembodiments, formulated in a pharmaceutically acceptable excipient, orintroduced in foods. In another embodiment, the invention provides foodspretreated or combined with a glutenase and methods for treating foodsto remove the toxic oligopeptides of gluten.

[0023] The methods of the invention can be used for prophylactic as wellas therapeutic purposes. As used herein, the term “treating” refers bothto the prevention of disease and the treatment of a disease or apre-existing condition. The invention provides a significant advance inthe treatment of ongoing disease, to stabilize or improve the clinicalsymptoms of the patient. Such treatment is desirably performed prior toloss of function in the affected tissues but can also help to restorelost function or prevent further loss of function. Evidence oftherapeutic effect may be any diminution in the severity of disease,particularly as measured by the severity of symptoms such as fatigue,chronic diarrhea, malabsorption of nutrients, weight loss, abdominaldistension, anemia, and other symptoms of Celiac Sprue. Other diseaseindicia include the presence of antibodies specific for glutens, thepresence of antibodies specific for tissue transglutaminase, thepresence of pro-inflammatory T cells and cytokines, damage to the villusstructure of the small intestine as evidenced by histological or otherexamination, enhanced intestinal permeability, and the like.

[0024] Patients that can benefit from the present invention may be ofany age and include adults and children. Children in particular benefitfrom prophylactic treatment, as prevention of early exposure to toxicgluten peptides can prevent initial development of the disease. Childrensuitable for prophylaxis can be identified by genetic testing forpredisposition, e.g. by HLA typing; by family history, by T cell assay,or by other medical means. As is known in the art, dosages may beadjusted for pediatric use.

[0025] Although the present invention is not to be bound by any theoryof action, it is believed that the primary event in Celiac Spruerequires certain gluten oligopeptides to access antigen binding siteswithin the lamina propria region interior to the relatively impermeablesurface intestinal epithelial layer. Ordinarily, oligopeptide endproducts of pancreatic protease processing are rapidly and efficientlyhydrolyzed into amino acids and/or di- or tri-peptides by gastricpeptidases before they are transported across the epithelial layer.However, glutens are particularly peptidase resistant, which may beattributed to the usually high proline content of these proteins, aresidue that is inaccessible to most gastric peptidases.

[0026] The normal assimilation of dietary proteins by the human gut canbe divided into three major phases: (i) initiation of proteolysis in thestomach by pepsin and highly efficient endo- and C-terminal cleavage inthe upper small intestine cavity (duodenum) by secreted pancreaticproteases and carboxypeptidases; (ii) further processing of theresulting oligopeptide fragments by exo- and endopeptidases anchored inthe brush border surface membrane of the upper small intestinalepithelium (jejunum); and (iii) facilitated transport of the resultingamino acids, di- and tripeptides across the epithelial cells into thelamina propria, from where these nutrients enter capillaries fordistribution throughout the body. Because most proteases and peptidasesnormally present in the human stomach and small intestine are unable tohydrolyze the amide bonds of proline residues, it is shown herein thatthe abundance of proline residues in gliadins and related proteins fromwheat, rye and barley can constitute a major digestive obstacle for theenzymes involved in phases (i) and (ii) above. This leads to anincreased concentration of relatively stable gluten derivedoligopeptides in the gut. Furthermore, because aminopeptidase andespecially carboxypeptidase activity towards oligopeptides with prolineresidues at the N- and C-termini, respectively, is low in the smallintestine, detoxification of gluten oligopeptides in phase (iii) aboveis also slow. By administering peptidases capable of cleaving suchgluten oligopeptides in accordance with the methods of the invention,the amount of toxic peptides is diminished, thereby slowing or blockingdisease progression.

[0027] Tissue transglutaminase (tTGase), an enzyme found on theextracellular surface in many organs including the intestine, catalyzesthe formation of isopeptide bonds between glutamine and lysine residuesof different polypeptides, leading to protein-protein crosslinks in theextracellular matrix. The enzyme tTGase is the primary focus of theautoantibody response in Celiac Sprue. Gliadins, secalins and hordeinscontain several sequences rich in Pro-Gin residues that arehigh-affinity substrates for tTGase; tTGase catalyzed deamidation of atleast some of these sequences dramatically increases their affinity forHLA-DQ2, the class II MHC allele present in >90% Celiac Sprue patients.Presentation of these deamidated epitopes by DQ2 positive antigenpresenting cells effectively stimulates proliferation ofgliadin-specific T cells from intestinal biopsies of most Celiac Spruepatients. The toxic effects of gluten include immunogenicity of thegluten oligopeptides, leading to inflammation; the lectin theorypredicts that gliadin peptides may also directly bind to surfacereceptors.

[0028] The present invention relates generally to methods and reagentsuseful in treating foodstuffs containing gluten with enzymes that digestthe oligopeptides toxic to Celiac Sprue patients. Although specificenzymes are exemplified herein, any of a number of alternative enzymesand methods apparent to those of skill in the art upon contemplation ofthis disclosure are equally applicable and suitable for use inpracticing the invention. The methods of the invention, as well as teststo determine their efficacy in a particular patient or application, canbe carried out in accordance with the teachings herein using proceduresstandard in the art. Thus, the practice of the present invention mayemploy conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology within the scope of those of skill in the art. Suchtechniques are explained fully in the literature, such as, “MolecularCloning: A Laboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction” (Mullis et al., eds., 1994); and “Current Protocols inImmunology” (J. E. Coligan et al., eds., 1991); as well as updated orrevised editions of all of the foregoing.

[0029] As used herein, the term “glutenase” refers to an enzyme usefulin the methods of the present invention that is capable, alone or incombination with endogenous or exogenously added enzymes, of cleavingtoxic oligopeptides of gluten proteins of wheat, barley, oats and ryeinto non-toxic fragments. Gluten is the protein fraction in cerealdough, which can be subdivided into glutenins and prolamines, which aresubclassified as gliadins, secalins, hordeins, and avenins from wheat,rye, barley and oat, respectively. For further discussion of glutenproteins, see the review by Wieser (1996) Acta Paediatr Suppl. 412:3-9,incorporated herein by reference.

[0030] In one embodiment, the term “glutenase” as used herein refers toa protease or a peptidase enzyme that meets one or more of the criteriaprovided herein. Using these criteria, one of skill in the art candetermine the suitability of a candidate enzyme for use in the methodsof the invention. Many enzymes will meet multiple criteria, includingtwo, three, four or more of the criteria, and some enzymes will meet allof the criteria. The terms “protease” or “peptidase” can refer to aglutenase and as used herein describe a protein or fragment thereof withthe capability of cleaving peptide bonds, where the scissile peptidebond may either be terminal or internal in oligopeptides or largerproteins. Prolyl-specific peptidases are glutenases useful in thepractice of the present invention.

[0031] Glutenases of the invention include protease and peptidaseenzymes having at least about 20% sequence identity at the amino acidlevel, more usually at least about 40% sequence identity, and preferablyat least about 70% sequence identity to one of the following peptidases:prolyl endopeptidase (PEP) from F. meningosepticum (Genbank accessionnumber D10980), PEP from A. hydrophila (Genbank accession numberD14005), PEP form S. caplsulata (Genbank accession number AB010298), DCPI from rabbit (Genbank accession number X62551), DPP IV from Aspergillusfumigatus (Genbank accession number U87950) or cysteine proteinase Bfrom Hordeum vulgare (Genbank accession number JQ1110).

[0032] In one embodiment of the present invention, the glutenase is aPEP. Homology-based identification (for example, by a PILEUP sequenceanalysis) of prolyl endopeptidases can be routinely performed by thoseof skill in the art upon contemplation of this disclosure to identifyPEPs suitable for use in the methods of the present invention. PEPs areproduced in microorganisms, plants and animals. PEPs belong to theserine protease superfamily of enzymes and have a conserved catalytictriad composed of a Ser, His, and Asp residues. Some of these homologshave been characterized, e.g. the enzymes from F. meningoscepticum,Aeromonas hydrophila, Aeromonas punctata, Novosphingobium capsulatum,Pyrococcus furiosus and from mammalian sources are biochemicallycharacterized PEPs. Others such as the Nostoc and Arabidopsis enzymesare likely to be PEPs but have not been fully characterized to date. Yetothers, such as the E. coli and M. xanthus enzymes, may not be PEPs butare homologous members of the serine protease superfamily, and can beuseful starting materials in protein engineering to make a PEP useful inthe practice of the present invention. Relative to the F.meningoscepticum enzyme, the pairwise sequence identity of this familyof enzymes is in the 30-60% range. Accordingly, PEPs include enzymeshaving >30% identity to the F. meningoscepticum enzyme (as in thePyrococcus enzymes), or having >40% identity (as in the Novosphingobiumenzymes), or having >50% identity (as in the Aeromonas enzymes) to theF. meningoscepticum enzyme.

[0033] A glutenase of the invention includes a peptidase or proteasethat has a specific activity of at least 2.5 U/mg, preferably 25 U/mgand more preferably 250 U/mg for cleavage of a peptide comprising one ofmore of the following motifs: Gly-Pro-pNA, Z-Gly-Pro-pNA (where Z is abenzyloxycarbonyl group), and Hip-His-Leu, where “Hip” is hippuric acid,pNA is para-nitroanilide, and 1 U is the amount of enzyme required tocatalyze the turnover of 1 μmole of substrate per minute.

[0034] A glutenase of the invention includes an enzyme belonging to anyof the following enzyme classifications: EC 3.4.21.26, EC 3.4.14.5, orEC 3.4.15.1.

[0035] A glutenase of the invention includes an enzyme having a kcat/Kmof at least about 2.5 s⁻¹ M⁻¹, usually at least about 250 s⁻¹ M⁻¹ andpreferably at least about 25000 s⁻¹ M⁻¹ for cleavage of any of thefollowing peptides under optimal conditions: (SEQ ID NO:1) QLQPFPQPQLPY,(SEQ ID NO:3) PQPQLPYPQPQLPY, (SEQ ID NO:13) QPQQSFPQQQ, (SEQ ID NO:14)QLQPFPQPELPY, (SEQ ID NO:15) PQPELPYPQPELPY, (SEQ ID NO:16) QPQQSFPEQQ.A glutenase of the invention includes peptidase or protease having aspecificity kcat/Km>2 mM⁻¹ s⁻¹ for the quenched fluorogenic substrateAbz-QPQQP-Tyr(NO₂)-D.

[0036] A glutenase useful in the practice of the present invention canbe identified by its ability to cleave a pretreated substrate to removetoxic gluten oligopeptides, where a “pretreated substrate” is a gliadin,hordein, secalin or avenin protein that has been treated withphysiological quantities of gastric and pancreatic proteases, includingpepsin (1:100 mass ratio), trypsin (1:100), chymotrypsin (1:100),elastase (1:500), and carboxypeptidases A and B (1:100). Pepsindigestion may be performed at pH 2 for 20 min., to mimic gastricdigestion, followed by further treatment of the reaction mixture withtrypsin, chymotrypsin, elastase and carboxypeptidase at pH 7 for 1 hour,to mimic duodenal digestion by secreted pancreatic enzymes. Thepretreated substrate comprises oligopeptides resistant to digestion,e.g. under physiological conditions.

[0037] The ability of a peptidase or protease to cleave a pretreatedsubstrate can be determined by measuring the ability of an enzyme toincrease the concentration of free NH₂-termini in a reaction mixturecontaining 1 mg/ml pretreated substrate and 10 μg/ml of the peptidase orprotease, incubated at 37° C. for 1 hour. A glutenase useful in thepractice of the present invention will increase the concentration of thefree amino termini under such conditions, usually by at least about 25%,more usually by at least about 50%, and preferably by at least about100%. A glutenase includes an enzyme capable of reducing the residualmolar concentration of oligopeptides greater than about 1000 Da in a 1mg/ml “pretreated substrate” after a 1 hour incubation with 10 μg/ml ofthe enzyme by at least about 2-fold, usually by at least about 5-fold,and preferably by at least about 10-fold. The concentration of sucholigopeptides can be estimated by methods known in the art, for examplesize exclusion chromatography and the like.

[0038] A glutenase of the invention includes an enzyme capable ofreducing the potency by which a “pretreated substrate” can antagonizebinding of (SEQ ID NO:17) PQPELPYPQPQLP to HLA-DQ2. The ability of asubstrate to bind to HLA-DQ is indicative of its toxicity; fragmentssmaller than about 8 amino acids are generally not stably bound to ClassII MHC. Treatment with a glutenase that digests toxic oligopeptides, byreducing the concentration of the toxic oligopeptides, prevents amixture containing them from competing with a test peptide for MHCbinding. To test whether a candidate glutenase can be used for purposesof the present invention, a 1 mg/ml solution of “pretreated substrate”may be first incubated with 10 μg/ml of the candidate glutenase, and theability of the resulting solution to displace radioactive (SEQ ID NO:18)PQPELPYPQPQPLP pre-bound to HLA-DQ2 molecules can then be quantified,with a reduction of displacement, relative to a non-treated control,indicative of utility in the methods of the present invention.

[0039] A glutenase of the invention includes an enzyme that reduces theanti-tTG antibody response to a “gluten challenge diet” in a CeliacSprue patient by at least about 2-fold, more usually by at least about5-fold, and preferably by at least about 10-fold. A “gluten challengediet” is defined as the intake of 100 g bread per day for 3 days by anadult Celiac Sprue patient previously on a gluten-free diet. Theanti-tTG antibody response can be measured in peripheral blood usingstandard clinical diagnostic procedures, as known in the art.

[0040] Excluded from the term “glutenase” are the following peptidases:human pepsin, human trypsin, human chymotrypsin, human elastase, papayapapain, and pineapple bromelain, and usually excluded are enzymes havinggreater than 98% sequence identity at the amino acid level to suchpeptidases, more usually excluded are enzymes having greater than 90%sequence identity at the amino acid level to such peptidases, andpreferably excluded are enzymes having greater than 70% sequenceidentity at the amino acid level to such peptidases.

[0041] Among gluten proteins with potential harmful effect to CeliacSprue patients are included the storage proteins of wheat, species ofwhich include Triticum aestivum; Triticum aethiopicum; Triticumbaeoticum; Triticum militinae; Triticum monococcum; Triticum sinskajae;Triticum timopheevii; Triticum turgidum; Triticum urartu, Triticumvavilovii; Triticum zhukovskyi; etc. A review of the genes encodingwheat storage proteins may be found in Colot (1990) Genet Eng (NY)12:225-41. Gliadin is the alcohol-soluble protein fraction of wheatgluten. Gliadins are typically rich in glutamine and proline,particularly in the N-terminal part. For example, the first 100 aminoacids of α- and γ-gliadins contain ˜35% and ˜20% of glutamine andproline residues, respectively. Many wheat gliadins have beencharacterized, and as there are many strains of wheat and other cereals,it is anticipated that many more sequences will be identified usingroutine methods of molecular biology. In one aspect of the presentinvention, genetically modified plants are provided that differ fromtheir naturally occurring counterparts by having gliadin proteins thatcontain a reduced content of glutamine and proline residues.

[0042] Examples of gliadin sequences include but are not limited towheat alpha gliadin sequences, for example as provided in Genbank,accession numbers AJ133612; AJ133611; AJ133610; AJ133609; AJ133608;AJ133607; AJ133606; AJ133605; AJ133604; AJ133603; AJ133602; D84341.1;U51307; U51306; U51304; U51303; U50984; and U08287. A sequence of wheatomega gliadin is set forth in Genbank accession number AF280605.

[0043] For the purposes of the present invention, toxic gliadinoligopeptides are peptides derived during normal human digestion ofgliadins and related storage proteins as described above, from dietarycereals, e.g. wheat, rye, barley, and the like. Such oligopeptides arebelieved to act as antigens for T cells in Celiac Sprue. For binding toClass II MHC proteins, immunogenic peptides are usually from about 8 to20 amino acids in length, more usually from about 10 to 18 amino acids.Such peptides may include PXP motifs, such as the motif PQPQLP (SEQ IDNO:8). Determination of whether an oligopeptide is immunogenic for aparticular patient is readily determined by standard T cell activationand other assays known to those of skill in the art.

[0044] As demonstrated herein, during digestion, peptidase resistantoligopeptides remain after exposure of glutens, e.g. gliadin, to normaldigestive enzymes. Examples of peptidase resistant oligopeptides areprovided, for example, as set forth in SEQ ID NO:5, 6, 7 and 10. Otherexamples of immunogenic gliadin oligopeptides are described in Wieser(1995) Baillieres Clin Gastroenterol 9(2):191-207, incorporated hereinby reference.

[0045] Determination of whether a candidate enzyme will digest a toxicgluten oligopeptide, as discussed above, can be empirically determined.For example, a candidate may be combined with an oligopeptide comprisingone or more Gly-Pro-pNA, Z-Gly-Pro-pNA, Hip-His-Leu,Abz-QLP-Tyr(NO₂)-PQ, Abz-PYPQPQ-Tyr(NO₂),PQP-Lys(Abz)-LP-Tyr(NO₂)-PQPQLP, PQPQLP-Tyr(NO₂)-PQP-Lys(Abz)-LP motifs;with one or more of the oligopeptides (SEQ ID NO:1) QLQPFPQPQLPY, (SEQID NO:3) PQPQLPYPQPQLPY, (SEQ ID NO:13) QPQQSFPQQQ, (SEQ ID NO:14)QLQPFPQPELPY, (SEQ ID NO:15) PQPELPYPQPELPY, (SEQ ID NO:16) QPQQSFPEQQor (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; or with apretreated substrate comprising one or more of gliadin, hordein, secalinor avenin proteins that have been treated with physiological quantitiesof gastric and pancreatic proteases. In each instance, the candidate isdetermined to be a glutenase of the invention if it is capable ofcleaving the oligopeptide. Glutenases that have a low toxicity for humancells and are active in the physiologic conditions present in theintestinal brush border are preferred for use in some applications ofthe invention, and therefore it may be useful to screen for suchproperties in candidate glutenases.

[0046] The oligopeptide or protein substrates for such assays may beprepared in accordance with conventional techniques, such as synthesis,recombinant techniques, isolation from natural sources, or the like. Forexample, solid-phase peptide synthesis involves the successive additionof amino acids to create a linear peptide chain (see Merrifield (1963)J. Am. Chem. Soc. 85:2149-2154). Recombinant DNA technology can also beused to produce the peptide.

[0047] Candidate glutenases for use in the practice of the presentinvention can be obtained from a wide variety of sources, includinglibraries of natural and synthetic proteins. For example, numerous meansare available for random and directed mutation of proteins.Alternatively, libraries of natural proteins in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Extracts of germinating wheat and other grasses is of interest as asource of candidate enzymes. Natural or synthetically produced librariesand compounds are readily modified through conventional chemical,physical and biochemical means, and such means can be used to producecombinatorial libraries. Known pharmacological agents may be subjectedto directed or random chemical modifications, such as acylation,alkylation, esterification, and amidification, to produce structuralanalogs of proteins.

[0048] Generally, a variety of assay mixtures are run in parallel withdifferent peptidase concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e. at zero concentration or below thelevel of detection. A variety of other reagents may be included in ascreening assay. These include reagents like salts, detergents, and thelike that are used to facilitate optimal activity and/or reducenon-specific or background interactions. Reagents that improve theefficiency of the assay may be used. The mixture of components is addedin any order that provides for the requisite activity. Incubations areperformed at any suitable temperature, typically between 4 and 40° C.Incubation periods are selected for optimum activity but can also beoptimized to facilitate rapid high-throughput screening or otherpurposes. Typically, between 0.1 and 1 hours will be sufficient.

[0049] The level of digestion of the toxic oligopeptide can be comparedto a baseline value. The disappearance of the starting material and/orthe presence of digestion products can be monitored by conventionalmethods. For example, a detectable marker can be conjugated to apeptide, and the change in molecular weight associated with the markeris then determined, e.g. acid precipitation, molecular weight exclusion,and the like. The baseline value can be a value for a control sample ora statistical value that is representative a control population. Variouscontrols can be conducted to ensure that an observed activity isauthentic, including running parallel reactions, positive and negativecontrols, dose response, and the like.

[0050] Active glutenases identified by the screening methods describedherein can serve as lead compounds for the synthesis of analog compoundsto identify glutenases with improved properties. Identification ofanalog compounds can be performed through use of techniques such asself-consistent field (SCF) analysis, configuration interaction (Cl)analysis, and normal mode dynamics analysis.

[0051] In one embodiment of the invention, the glutenase is a prolylendopeptidase (PEP, EC 3.4.21.26). Prolyl endopeptidases are widelydistributed in microorganisms, plants and animals, and have been clonedfrom Flavobacterium meningosepticum, (Yoshimoto et al. (1991) J.Biochem. 110, 873-8); Aeromonas hydrophyla (Kanatani et al. (1993) J.Biochem. 113, 790-6); Sphingomonas capsulata (Kabashima et al. (1998)Arch. Biochem. Biophys. 358, 141-148), Pyrococcus furious (Robinson etal. (1995) Gene 152, 103-6); pig (Rennex et al. (1991) Biochemistry 30,2195-2030); and the like. The suitability of a particular enzyme isreadily determined by the assays described above, by clinical testing,determination of stability in formulations, and the like. Other sourcesof PEP include Lactobacilli (Habibi-Najafi et al. (1994) J. Dairy Sci.77, 385-392), from where the gene of interest can be readily clonedbased on sequence homology to the above PEP's or via standard reversegenetic procedures involving purification, amino-acid sequencing,reverse translation, and cloning of the gene encoding the targetextracellular enzyme.

[0052] In another embodiment of the invention, glutenases are peptidasespresent in the brush border, which are supplemented. Formulations ofinterest may comprise such enzymes in combination with other peptidases.Peptidases present in brush border include dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), and dipeptidyl carboxypeptidase (DCP, EC 3.4.15.1).The human form of these proteins may be used, or modified forms may beisolated from other suitable sources. Example of DPP IV enzymes includeAspergillus spp. (e.g. Byun et al. (2001) J. Agric. Food Chem. 49,2061-2063), ruminant bacteria such as Prevotella albensis M384 (NCBIprotein database Locus # CAC42932), dental bacteria such asPorphyromonas gingivalis W83 (Kumugai et al. (2000) Infect. Immun. 68,716-724), lactobacilli such as Lactobacillus helveticus (e.g. Vesanto,et al, (1995) Microbiol. 141, 3067-3075), and Lactococcus lactis (Mayoet al., (1991) Appl. Environ. Microbiol. 57, 38-44). Other DPP IVcandidates can readily be recognized based on homology to the aboveenzymes, preferably >30% sequence identity. Similarly, secreteddipeptidyl carboxypeptidases that cleave C-terminal X-Pro sequences arefound in many microbial sources including Pseudomonas spp (e.g.Ogasawara et al, (1997) Biosci. Biotechnol. Biochem. 61, 858-863),Streptomyces spp. (e.g. Miyoshi et al., (1992) J. Biochem. 112, 253-257)and Aspergilli spp. (e.g. Ichishima et al., (1977) J. Biochem. 81,1733-1737). Of particular interest is the enzyme from Aspergillus saitoi(Ichishima), due to its high activity at acidic pH values. Although thegenes encoding many of these enzymes have not yet been cloned, they canbe readily cloned by standard reverse genetic procedures. The DCP Ienzymes can be purified from the extracellular medium based on theirability to hydrolyze (SEQ ID NO:19) Z-Gly-Pro-Leu-Gly-Pro, Z-Gly-Pro, orHip-Gly-Pro. Alternatively, putative DCP I genes can be identified basedon homology to the E. coli enzyme (NCBI protein database LocusCAA41014.)

[0053] In another embodiment of the invention, glutenases areendoproteases found in developing grains of toxic cereals such as wheat,barley and rye. For example, Dominguez and Cejudo (Plant Physiol. 112,1211-1217, 1996) have shown that the endosperm of wheat (i.e. the partof the grain that contains gliadin and glutenin) contains a variety ofneutral and acid proteases. Although these proteases have not beenindividually characterized, they are expected to be an especially richsource of glutenases. Moreover, although the genes encoding theseproteases have not yet been cloned, Dominguez and Cejudo haveestablished a convenient SDS-PAGE assay for identification andseparation of these proteases. After excision of the correspondingprotein bands from the gel, limited sequence information can beobtained. The cDNA encoding these proteases can therefore be readilycloned from this information using established reverse geneticprocedures, and expressed in heterologous bacterial or fungal hosts. Ofparticular interest are proteases that hydrolyze α2-gliadin within the33-mer amino acid sequence identified in Example 2 below. Of furtherinterest are the subset of these proteases that retain activity atacidic pH values (pH2-5) encountered in the stomach.

[0054] The amino acid sequence of a glutenase, e.g. a naturallyoccurring glutenase, can be altered in various ways known in the art togenerate targeted changes in sequence and additional glutenase enzymesuseful in the formulations and compositions of the invention. Suchvariants will typically be functionally-preserved variants, whichdiffer, usually in sequence, from the corresponding native or parentprotein but still retain the desired biological activity. Variants alsoinclude fragments of a glutenase that retain enzymatic activity. Variousmethods known in the art can be used to generate targeted changes, e.g.phage display in combination with random and targeted mutations,introduction of scanning mutations, and the like.

[0055] A variant can be substantially similar to a native sequence, i.e.differing by at least one amino acid, and can differ by at least two butusually not more than about ten amino acids (the number of differencesdepending on the size of the native sequence). The sequence changes maybe substitutions, insertions or deletions. Scanning mutations thatsystematically introduce alanine, or other residues, may be used todetermine key amino acids. Conservative amino acid substitutionstypically include substitutions within the following groups: (glycine,alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid);(asparagine, glutamine); (serine, threonine); (lysine, arginine); and(phenylalanine, tyrosine).

[0056] Glutenase fragments of interest include fragments of at leastabout 20 contiguous amino acids, more usually at least about 50contiguous amino acids, and may comprise 100 or more amino acids, up tothe complete protein, and may extend further to comprise additionalsequences. In each case, the key criterion is whether the fragmentretains the ability to digest the toxic oligopeptides that contribute tothe symptoms of Celiac Sprue.

[0057] Modifications of interest that do not alter primary sequenceinclude chemical derivatization of proteins, e.g., acetylation orcarboxylation. Also included are modifications of glycosylation, e.g.those made by modifying the glycosylation patterns of a protein duringits synthesis and processing or in further processing steps; e.g. byexposing the protein to enzymes that affect glycosylation, such asmammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

[0058] Also useful in the practice of the present invention are proteinsthat have been modified using molecular biological techniques and/orchemistry so as to improve their resistance to proteolytic degradationand/or to acidic conditions such as those found in the stomach, and tooptimize solubility properties or to render them more suitable as atherapeutic agent. For example, the backbone of the peptidase can becyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem.275:23783-23789). Analogs of such proteins include those containingresidues other than naturally occurring L-amino acids, e.g. D-aminoacids or non-naturally occurring synthetic amino acids.

[0059] The glutenase proteins of the present invention may be preparedby in vitro synthesis, using conventional methods as known in the art.Various commercial synthetic apparatuses are available, for example,automated synthesizers by Applied Biosystems, Inc., Foster City, Calif.,Beckman, and other manufacturers. Using synthesizers, one can readilysubstitute for the naturally occurring amino acids one or more unnaturalamino acids. The particular sequence and the manner of preparation willbe determined by convenience, economics, purity required, and the like.If desired, various groups can be introduced into the protein duringsynthesis that allow for linking to other molecules or to a surface. Forexample, cysteines can be used to make thioethers, histidines can beused for linking to a metal ion complex, carboxyl groups can be used forforming amides or esters, amino groups can be used for forming amides,and the like.

[0060] The glutenase proteins useful in the practice of the presentinvention may also be isolated and purified in accordance withconventional methods from recombinant production systems and fromnatural sources. A lysate can be prepared from the expression host andthe lysate purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, and/or other purificationtechniques. Typically, the compositions used in the practice of theinvention will comprise at least 20% by weight of the desired product,more usually at least about 75% by weight, preferably at least about 95%by weight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein.

[0061] In one aspect, the present invention provides a purifiedpreparation of a glutenase. Prior to the present invention, there was noneed for a glutenase that could be ingested by a human or mixed with afoodstuff. Thus, prior to the present invention most glutenases did notexist in a form free of contaminants that could be deleterious to ahuman if ingested. The present invention creates a need for suchglutenase preparations and provides them and methods for preparing them.In a related embodiment, the present invention provides novel foodstuffsthat are derived from gluten-containing foodstuffs but have been treatedto reduce the concentration and amount of the oligopeptides andoligopeptide sequences discovered to be toxic to Celiac Sprue patients.While gluten-free or reduced-gluten content foods have been made, thefoodstuffs of the present invention differ from such foodstuffs not onlyby the manner in which they are prepared, by treatment of the foodstuffwith a glutenase, but also by their content, as the methods of the priorart result in alteration of non-toxic (to Celiac Sprue patients)components of the foodstuff, resulting in a different taste andcomposition. Prior art foodstuffs include, for example, CodexAlimentarius wheat starch, which is available in Europe and has <100 ppmgluten. The starch is usually prepared by processes that take advantageof the fact that gluten is insoluble in water whereas starch is soluble.

[0062] In one embodiment of the present invention, a Celiac Spruepatient is, in addition to being provided a glutenase or food treated inaccordance with the present methods, provided an inhibitor of tissuetransglutaminase, an anti-inflammatory agent, an anti-ulcer agent, amast cell-stabilizing agents, and/or and an-allergy agent. Examples ofsuch agents include HMG-CoA reductase inhibitors with anti-inflammatoryproperties such as compactin, lovastatin, simvastatin, pravastatin andatorvastatin; anti-allergic histamine H1 receptor antagonists such asacrivastine, cetirizine, desloratadine, ebastine, fexofenadine,levocetirizine, loratadine and mizolastine; leukotriene receptorantagonists such as montelukast and zafirlukast; COX2 inhibitors such ascelecoxib and rofecoxib; p38 MAP kinase inhibitors such as BIRB-796; andmast cell stabilizing agents such as sodium chromoglycate (chromolyn),pemirolast, proxicromil, repirinast, doxantrazole, amlexanox nedocromiland probicromil.

[0063] As used herein, compounds which are “commercially available” maybe obtained from commercial sources including but not limited to AcrosOrganics (Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wis., includingSigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), AvocadoResearch (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet(Cornwall, U.K.), Chemservice Inc. (West Chester Pa.), Crescent ChemicalCo. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman Kodak Company(Rochester N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), FisonsChemicals (Leicestershire UK), Frontier Scientific (Logan Utah), ICNBiomedicals, Inc. (Costa Mesa Calif.), Key Organics (Cornwall U.K.),Lancaster Synthesis (Windham N.H.), Maybridge Chemical Co. Ltd.(Cornwall U.K.), Parish Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc.(Waterbury Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co.(Rockford Ill.), Riedel de Haen AG (Hannover, Germany), Spectrum QualityProduct, Inc. (New Brunswick, N.J.), TCI America (Portland Oreg.), TransWorld Chemicals, Inc. (Rockville Md.), Wako Chemicals USA, Inc.(Richmond Va.), Novabiochem and Argonaut Technology.

[0064] Compounds useful for co-administration with the glutenases andtreated foodstuffs of the invention can also be made by methods known toone of ordinary skill in the art. As used herein, “methods known to oneof ordinary skill in the art” may be identified though various referencebooks and databases. Suitable reference books and treatises that detailthe synthesis of reactants useful in the preparation of compounds of thepresent invention, or provide references to articles that describe thepreparation, include for example, “Synthetic Organic Chemistry”, JohnWiley & Sons, Inc., New York; S. R. Sandler et al., “Organic FunctionalGroup Preparations,” 2nd Ed., Academic Press, New York, 1983; H. O.House, “Modern Synthetic Reactions”, 2nd Ed., W. A.

[0065] Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist,“Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J.March, “Advanced Organic Chemistry: Reactions, Mechanisms andStructure”, 4th Ed., Wiley-Interscience, New York, 1992. Specific andanalogous reactants may also be identified through the indices of knownchemicals prepared by the Chemical Abstract Service of the AmericanChemical Society, which are available in most public and universitylibraries, as well as through on-line databases (the American ChemicalSociety, Washington, D.C., www.acs.org may be contacted for moredetails). Chemicals that are known but not commercially available incatalogs may be prepared by custom chemical synthesis houses, where manyof the standard chemical supply houses (e.g., those listed above)provide custom synthesis services.

[0066] The glutenase proteins of the invention and/or the compoundsadministered therewith are incorporated into a variety of formulationsfor therapeutic administration. In one aspect, the agents are formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and are formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.As such, administration of the glutenase and/or other compounds can beachieved in various ways, usually by oral administration. The glutenaseand/or other compounds may be systemic after administration or may belocalized by virtue of the formulation, or by the use of an implant thatacts to retain the active dose at the site of implantation.

[0067] In pharmaceutical dosage forms, the glutenase and/or othercompounds may be administered in the form of their pharmaceuticallyacceptable salts, or they may also be used alone or in appropriateassociation, as well as in combination with other pharmaceuticallyactive compounds. The agents may be combined, as previously described,to provide a cocktail of activities. The following methods andexcipients are exemplary and are not to be construed as limiting theinvention.

[0068] For oral preparations, the agents can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0069] In one embodiment of the invention, the oral formulationscomprise enteric coatings, so that the active agent is delivered to theintestinal tract. Enteric formulations are often used to protect anactive ingredient from the strongly acid contents of the stomach. Suchformulations are created by coating a solid dosage form with a film of apolymer that is insoluble in acid environments, and soluble in basicenvironments. Exemplary films are cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methylcellulose phthalate andhydroxypropyl methylcellulose acetate succinate, methacrylatecopolymers, and cellulose acetate phthalate.

[0070] Other enteric formulations comprise engineered polymermicrospheres made of biologically erodable polymers, which displaystrong adhesive interactions with gastrointestinal mucus and cellularlinings and can traverse both the mucosal absorptive epithelium and thefollicle-associated epithelium covering the lymphoid tissue of Peyer'spatches. The polymers maintain contact with intestinal epithelium forextended periods of time and actually penetrate it, through and betweencells. See, for example, Mathiowitz et al. (1997) Nature 386 (6623):410-414. Drug delivery systems can also utilize a core of superporoushydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh etal. (2001) J Control Release 71(3):307-18.

[0071] In another embodiment, a microorganism, for example bacterial oryeast culture, capable of producing glutenase is administered to apatient. Such a culture may be formulated as an enteric capsule; forexample, see U.S. Pat. No. 6,008,027, incorporated herein by reference.Alternatively, microorganisms stable to stomach acidity can beadministered in a capsule, or admixed with food preparations.

[0072] In another embodiment, the glutenase is admixed with food, orused to pre-treat foodstuffs containing glutens. Glutenase present infoods can be enzymatically active prior to or during ingestion, and maybe encapsulated or otherwise treated to control the timing of activity.Alternatively, the glutenase may be encapsulated to achieve a timedrelease after ingestion, e.g. in the intestinal tract.

[0073] Formulations are typically provided in a unit dosage form, wherethe term “unit dosage form,” refers to physically discrete unitssuitable as unitary dosages for human subjects, each unit containing apredetermined quantity of glutenase in an amount calculated sufficientto produce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for the unitdosage forms of the present invention depend on the particular complexemployed and the effect to be achieved, and the pharmacodynamicsassociated with each complex in the host.

[0074] The pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are commercially available. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are commercially available. Any compound useful inthe methods and compositions of the invention can be provided as apharmaceutically acceptable base addition salt. “Pharmaceuticallyacceptable base addition salt” refers to those salts which retain thebiological effectiveness and properties of the free acids, which are notbiologically or otherwise undesirable. These salts are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, thesodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Preferred inorganicsalts are the ammonium, sodium, potassium, calcium, and magnesium salts.Salts derived from organic bases include, but are not limited to, saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like.Particularly preferred organic bases are isopropylamine, diethylamine,ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0075] Depending on the patient and condition being treated and on theadministration route, the glutenase may be administered in dosages of0.01 mg to 500 mg/kg body weight per day, e.g. about 20 mg/day for anaverage person. A typical dose of glutenase in patients will be in atleast about 1 mg/adult, more usually at least about 10 mg; andpreferably at least about 50 mg; usually not more than about 5 g, moreusually not more than about 1 g, and preferably not more than about 500mg. Dosages will be appropriately adjusted for pediatric formulation. Inchildren the effective dose may be lower, for example at least about 0.1mg, or 0.5 mg. In combination therapy involving, for example, a PEP+DPPIV or PEP+DCP I, a comparable dose of the two enzymes may be given;however, the ratio will be influenced by the relative stability of thetwo enzymes toward gastric and duodenal inactivation.

[0076] Those of skill will readily appreciate that dose levels can varyas a function of the specific enzyme, the severity of the symptoms andthe susceptibility of the subject to side effects. Some of theglutenases are more potent than others. Preferred dosages for a givenenzyme are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

[0077] Other formulations of interest include formulations of DNAencoding glutenases of interest, so as to target intestinal cells forgenetic modification. For example, see U.S. Pat. No. 6,258,789, hereinincorporated by reference, which discloses the genetic alteration ofintestinal epithelial cells.

[0078] The methods of the invention are used to treat foods to beconsumed or that are consumed by individuals suffering from Celiac Sprueand/or dermatitis herpetiformis by delivering an effective dose ofglutenase. If the glutenase is administered directly to a human, thenthe active agent(s) are contained in a pharmaceutical formulation.Alternatively, the desired effects can be obtained by incorporatingglutenase into food products or by administering live organisms thatexpress glutenase, and the like. Diagnosis of suitable patients mayutilize a variety of criteria known to those of skill in the art. Aquantitative increase in antibodies specific for gliadin, and/or tissuetransglutaminase is indicative of the disease. Family histories and thepresence of the HLA alleles HLA-DQ2 [DQ(a1*0501, b1*02)] and/or DQ8[DQ(a1*0301, b1*0302)] are indicative of a susceptibility to thedisease.

[0079] The therapeutic effect can be measured in terms of clinicaloutcome or can be determined by immunological or biochemical tests.Suppression of the deleterious T-cell activity can be measured byenumeration of reactive Th1 cells, by quantitating the release ofcytokines at the sites of lesions, or using other assays for thepresence of autoimmune T cells known in the art. Alternatively, one canlook for a reduction in symptoms of a disease.

[0080] Various methods for administration may be employed, preferablyusing oral administration, for example with meals. The dosage of thetherapeutic formulation will vary widely, depending upon the nature ofthe disease, the frequency of administration, the manner ofadministration, the clearance of the agent from the host, and the like.The initial dose can be larger, followed by smaller maintenance doses.The dose can be administered as infrequently as weekly or biweekly, ormore often fractionated into smaller doses and administered daily, withmeals, semi-weekly, or otherwise as needed to maintain an effectivedosage level.

[0081] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of the invention or to represent that the experiments beloware all or the only experiments performed. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,temperature, and the like), but some experimental errors and deviationsmay be present. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1 Detection of Immunodominant Peptides from Gliadin and Enzymesthat Degrade Them

[0082] The following examples describe the discovery andcharacterization of a small number of immunodominant peptides fromgliadin, which account for most of the stimulatory activity of dietarygluten on intestinal and peripheral T lymphocytes found in Celiac Spruepatients. The proteolytic kinetics of these immunodominant peptides wereanalyzed at the small intestinal surface. Brush border membrane vesiclesfrom adult rat intestines were used to show that theseproline-glutamine-rich peptides are exceptionally resistant to enzymaticprocessing, and that dipeptidyl peptidase IV and dipeptidylcarboxypeptidase are the rate-limiting enzymes in their digestion.Supplementation of the brush border membrane with trace quantities of abacterial prolyl endopeptidase leads to rapid destruction of thesegliadin peptides. These results provide the basis for enzyme-mediatedtherapies for treating food for provision to Celiac Sprue patients, andfor treating such patients directly that offer distinct advantages overthe only current therapeutic option, which is strict exclusion of glutencontaining food.

[0083] To investigate the digestion of gluten, liquid chromatographycoupled mass spectroscopy analysis (LC-MS-MS) was utilized toinvestigate the pathways and associated kinetics of hydrolysis ofimmunodominant gliadin peptides treated with rat BBM preparations.Because the rodent is an excellent small animal model for humanintestinal structure and function, rat BBM was chosen as a suitablemodel system for these studies.

[0084] BBM fractions were prepared from rat small intestinal mucosa asdescribed in Ahnen et al. (1982) J. Biol. Chem. 257, 12129-35. Thespecific activities of the known BB peptidases were determined to be 127μU/μg for Aminopeptidase N (APN, EC 3.4.11.2), 60 μU/μg for dipeptidylpeptidase IV (DPP IV, EC 3.4.14.5), and 41 μU/μg for dipeptidylcarboxypeptidase (DCP, EC 3.4.15.1) using standard assays. No prolineaminopeptidase (EC 3.4.11.5) or prolyl endopeptidase activity (PEP, EC3.4.21.26) activity was detectable (<5 μU/μg). Alkaline phosphatase andsucrase were used as control BBM enzymes with activities of 66 μU/μg and350 μU/μg, respectively.

[0085] BBM fractions were partially purified from the small intestinalmucosa of adult female rats maintained on an ad libitum diet ofwheat-based standard rodent chow. Total protein content was determinedby a modified method of Lowry with BSA as a standard. Alkalinephosphatase activity was determined with nitrophenyl phosphate. Sucraseactivity was measured using a coupled glucose assay. DPP IV, prolineaminopeptidase and APN were assayed continuously at 30° C. in 0.1MTris-HCl, pH 8.0, containing 1 mM of the p-nitroanilides (ε=8,800 M⁻¹cm⁻¹) Gly-Pro-pNA, Pro-pNA or Leu-pNA, the latter in additional 1% DMSOto improve solubility. DCP activity was measured in a 100 μl reaction asthe release of hippuric acid from Hip-His-Leu. PEP activity wasdetermined continuously with 0.4 mM Z-Gly-Pro-pNA in PBS:H₂O:dioxane(8:1.2:0.8) at 30° C. One unit is the consumption of 1 μmol substrateper minute.

[0086] DPP IV and DCP are both up-regulated by a high proline content inthe diet. However, APN activity using standard substrates was found tobe higher than DPP IV even when fed extreme proline rich diets. Also,although a higher DCP vs. CPP activity has been observed with the modelpeptide Z-GPLAP at saturating concentrations, a difference in Km valuescould easily account the reversed ratio measured. The amount of 100 μMwas chosen as the initial peptide concentration, because non-saturatingkinetics (k_(cat)/K_(m)) were considered to be physiologically morerelevant than the maximal rates of hydrolysis (k_(cat)).

[0087] Proteolysis with the BBM preparation was investigated using thepeptide (SEQ ID NO:1) QLQPFPQPQLPY, a product of chymotryptic digestionof α-9 gliadin (Arentz-Hansen et al. (2000) J. Exp. Med. 191, 603-12).This peptide has been shown to stimulate proliferation of T cellsisolated from most Celiac Sprue patients, and hence is considered topossess an immunodominant epitope. It was subjected to BBM digestion,followed by LC-MS-MS analysis. A standard 50 μl digestion mixturecontained 100 μM of synthetic peptide, 10 μM tryptophan andCbz-tryptophan as internal standards, and resuspended BBM preparationswith a final protein content of 27 ng/μl and exogenous proteins, asindicated, in phosphate buffered saline. After incubation at 37° C. forthe indicated time, the enzymes were inactivated by heating to 95° C.for 3 minutes. The reaction mixtures were analyzed by LC-MS(SpectraSystem, ThermoFinnigan) using a C18 reversed phase column (Vydac218TP5215, 2.1×150 mm) with water:acetonitrile:formic acid(0.1%):trifluoroacetic acid (0.025%) as the mobile phase (flow: 0.2ml/min) and a gradient of 10% acetonitrile for 3 minutes, 10-20% for 3minutes, 20-25% for 21 minutes followed by a 95% wash. Peptide fragmentsin the mass range of m/z=300-2000 were detected by electrosprayionization mass spectroscopy using a LCQ ion trap and their identitieswere confirmed by MSMS fragmentation patterns.

[0088] While the parent peptide (SEQ ID NO:1) QLQPFPQPQLPY disappearedwith an apparent half life of 35 min, several intermediates wereobserved to accumulate over prolonged periods (FIG. 1A). The MSintensities (m/z=300-2000 g/mol) and UV₂₈₀ absorbances of the parentpeptides (SEQ ID NO:1) QLQPFPQPQLPY and (SEQ ID NO:3) PQPQLPYPQPQLPYwere found to depend linearly on concentration in the range of 6-100 μM.The reference peptides (SEQ ID NO:4) PQPQLPYPQPQLP, (SEQ ID NO:5)QLQPFPQPQLP, (SEQ ID NO:6) QPQFPQPQLPY and (SEQ ID NO:7) QPFPQPQLP weregenerated individually by limited proteolysis of the parent peptideswith 10 μg/ml carboxypeptidase A (C-0261, Sigma) and/or 5.9 μg/mlleucine aminopeptidase (L-5006, Sigma) for 160 min at 37° C. andanalyzed by LC-MS as in FIG. 1.

[0089] Indeed, the subsequent processing of the peptide wassubstantially retarded (FIG. 1B). The identities of the majorintermediates were confirmed by tandem MS, and suggested an unusuallyhigh degree of stability of the (SEQ ID NO:8) PQPQLP sequence, a commonmotif in T cell stimulating peptides. Based on this data and the knownamino acid preferences of the BBM peptidases, the digestive breakdown of(SEQ ID NO:1) QLQPFPQPQLPY was reconstructed, as shown in the insert ofFIG. 1B. The preferred pathway involves serial cleavage of theN-terminal glutamine and leucine residues by aminopeptidase N (APN),followed by removal of the C-terminal tyrosine by carboxypeptidase P(CPP) and hydrolysis of the remaining N-terminal QP-dipeptide by DPP IV.As seen in FIG. 1B, the intermediate (SEQ ID NO:6) QPFPQPQLPY (formed byAPN attack on the first two N-terminal residues) and its derivatives areincreasingly resistant to further hydrolysis. Because the high prolinecontent seemed to be a major cause for this proteolytic resistance,digestion was compared with a commercially available non-proline controlpeptide (SEQ ID NO:9) RRLIEDNEYTARG (Sigma, St. Louis, Mo.). Initialhydrolysis was much faster (t_(1/2)=10 min). More importantly, digestiveintermediates were only transiently observed and cleared completelywithin one hour, reflecting a continuing high specificity of the BBM forthe intermediate peptides.

[0090] Because the three major intermediate products (SEQ ID NO:10)QPFPQPQLPY, (SEQ ID NO:7) QPFPQPQLP, (SEQ ID NO:11) FPQPQLP) observedduring BBM mediated digestion of (SEQ ID NO:1) QLQPFPQPQLPY aresubstrates for DPP IV, the experiment was repeated in the presence of a6-fold excess activity of exogenous fungal DPP IV. Whereas therelatively rapid decrease of the parent peptide and the intermediatelevels of (SEQ ID NO:5) QLQPFPQPQLP were largely unchanged, theaccumulation of DPP IV substrates was entirely suppressed, and completedigestion was observed within four hours. (FIG. 1B, open bars).

[0091] To investigate the rate-limiting steps in BBM mediated digestionof gliadin peptides from the C-terminal end, another knownimmunodominant peptide derived from wheat α-gliadin, (SEQ ID NO:3)PQPQLPYPQPQLPY, was used. Although peptides with N-terminal prolineresidues are unlikely to form in the small intestine (none were observedduring BBM digestion of (SEQ ID NO:1) QLQPFPQPQLPY, FIG. 1A), they serveas a useful model for the analysis of C-terminal processing, because theN-terminal end of this peptide can be considered proteolyticallyinaccessible due to minimal proline aminopeptidase activity in the BBM.As shown in FIG. 2, this peptide is even more stable than (SEQ ID NO:1)QLQPFPQPQLPY. In particular, removal of the C-terminal tyrosine residueby carboxypeptidase P (CPP) is the first event in its breakdown, andmore than four times slower than APN activity on (SEQ ID NO:1)QLQPFPQPQLPY (FIG. 1B). The DCP substrate (SEQ ID NO:4) PQPQLPYPQPQLPemerges as a major intermediate following carboxypeptidase P ctgalysis,and is highly resistant to further digestion, presumably due to the lowlevel of endogenous DCP activity naturally associated with the BBM. Toconfirm the role of DCP as a rate-limiting enzyme in the C-terminalprocessing of immunodominant gliadin peptides, the reaction mixtureswere supplemented with rabbit lung DCP. Exogenous DCP significantlyreduced the accumulation of (SEQ ID NO:4) PQPQLPYPQPQLP after overnightincubation in a dose dependent manner. Conversely, the amount ofaccumulated (SEQ ID NO:4) PQPQLPYPQPQLP increased more than 2-fold inthe presence of 10 μM of captopril, a DCP-specific inhibitor, ascompared with unsupplemented BBM.

[0092] Together, the above results demonstrate that (i) immunodominantgliadin peptides are exceptionally stable toward breakdown catalyzed byBBM peptidases, and (ii) DPP IV and especially DCP are rate-limitingsteps in this breakdown process at the N- and C-terminal ends of thepeptides, respectively. Because BBM exopeptidases are restricted to N-or C-terminal processing, it was investigated if generation ofadditional free peptide ends by pancreatic enzymes would acceleratedigestion. Of the pancreatic proteases tested, only elastase at a high(non-physiological) concentration of 100 ng/μl was capable ofhydrolyzing (SEQ ID NO:3) PQPQLPYPQPQ^(↓)LPY. No proteolysis wasdetected with trypsin or chymotrypsin.

[0093] Alerted by the high proline content as a hallmark of mostimmunogenic gliadin peptides, a proline-specific endopeptidase wastested for the generation of new, free peptide termini. A literaturesearch on available proteases led to the identification of prolylendopeptidase (PEP) from Flavobacterium meningosepticum, which isspecific for the C-terminal cleavage of prolines and readily availablefrom recombinant sources (Yoshimoto et al. (1991) J. Biochem. 110,873-8). The stable (SEQ ID NO:4) PQPQLPYPQPQLP intermediate was digestedwith BBM in the presence of exogenous PEP. FIG. 3 shows the dosedependent acceleration of (SEQ ID NO:4) PQPQLPYPQPQLP digestion withincreasing PEP concentration. As little as 3.5 pg PEP/27 ng BBM proteinwas sufficient to double the extent of proteolysis of this gliadinfragment compared to incubation with BBM alone. In comparison, othercommonly used proteases like papain, bromelain or porcine elastase weremuch less efficient, requiring 30-fold (papain) or 3000-fold (bromelain,elastase) higher amounts of enzyme compared to PEP to give similarresults. Their proteolysis was restricted to the cleavage of theGln⁴-Leu⁵ and/or Gln¹¹-Leu¹² bonds.

[0094] Prolyl endopeptidase (EC 3.4.21.26) had a preference for thePro⁸-Gln⁹ and to a lesser extent the Pro⁶-Tyr⁷ bond of the (SEQ ID NO:4)PQPQLP^(↓)YP^(↓)QPQLP peptide. A similar preferential cleavage was foundfor (SEQ ID NO:1) QLQPFP^(↓)QPQLPY. This is in agreement with thepreference of this prolyl endopeptidase for a second proline in the S2′position (Bordusa and Jakubke (1998) Bioorg. Med. Chem. 6, 1775-80).Based on this P^(↓)XP motif and on the present data, up to 16 new, majorcleavage sites can be predicted in the α2-gliadin sequence, a majorsource of immunodominant epitopes identified thus far upon PEPtreatment. All of them are located in the critical N-terminal part. Theinternal cleavage by PEP can be expected to generate additional(otherwise inaccessible) substrates for DPP IV and DCP therebycomplementing the natural assimilation process of gliadins by the BBM.Thus, the specificity of prolyl endopeptidase is ideally suited fordetoxification of persistent immunoactive gliadin peptides in CeliacSprue.

[0095] The above data demonstrates that proline-rich gliadin peptidesare extraordinarily resistant to digestion by small intestinal endo- andexopeptidases, and therefore are likely to accumulate at highconcentrations in the intestinal cavity after a gluten rich meal. Thepathological implication of digestive resistance is strengthened by theobserved close correlation of proline content and celiac toxicity asobserved in the various common cereals (Schuppan (2000) Gastroenterology119, 234-42). This analysis of the digestive pathways of immunodominantpeptides also provides a mechanism for determining whether enzymescapable of accelerating this exceptionally slow process can betherapeutically useful in the Celiac Sprue diet.

[0096] Addition of exogenous DPP IV and DCP can compensate for theintrinsically slow proline processing by the BBM, although both enzymesrely on efficient generation of free N- and C-termini by endoproteolyticcleavage. In a preferred embodiment, a soluble bacterial prolylendopeptidase (PEP) is used, which was shown to be extremely efficientat hydrolyzing the proline-rich gliadin fragments. Although PEP isexpressed in human brain, lung, kidney and intestine, no such activityhas been reported in the brush border.

[0097] Supplementation of the Celiac Sprue diet with bioavailable PEP(with or without DPP IV and/or DCP), by virtue of facilitating gliadinpeptide cleavage to non-toxic and/or digestible fragments, is useful inattenuating or eliminating the inflammatory response to gluten. Such atreatment regimen is analogous to the enzyme therapy treatment used totreat lactose intolerance, where orally administered lactase iseffective in cleaving and thereby detoxifying the lactose in milkproducts. Prolyl endopeptidases are widely distributed inmicroorganisms, plants and animals and have been cloned from Aeromonashydrophyla (Kanatani et al. (1993) J. Biochem. 113, 790-6); Pyrococcusfurious (Robinson et al. (1995) Gene 152, 103-6) and from pig brain(Rennex et al. (1991) Biochemistry 30, 2195-2030). These isozymesconstitute alternative detoxifying peptidases. Furthermore, the prolylendopeptidase used in this study is readily amenable to proteinengineering by directed evolution. Thus, optimization of PEP specificitytowards immunogenic gliadin peptides can be achieved.

EXAMPLE 2 Further Characterization of Immunodominant Gliadin Peptidesand Means for Their Digestion

[0098] It has long been known that the principal toxic components ofwheat gluten are a family of closely related Pro-Gln rich proteinscalled gliadins. Peptides from a short segment of α-gliadin appear toaccount for most of the gluten-specific recognition by CD4+ T cells fromCeliac Sprue patients. These peptides are substrates of tissuetransglutaminase (tTGase), the primary auto-antigen in Celiac Sprue, andthe products of this enzymatic reaction bind to the class II HLA DQ2molecule. This example describes a combination of in vitro and in vivoanimal and human studies used to characterize this “immunodominant”region of α-gliadin as part of an unusually long proteolytic productgenerated by the digestive process that: (a) is exceptionally resistantto further breakdown by gastric, pancreatic and intestinal brush borderproteases; (b) is the highest specificity substrate of human tissuetransglutaminase (tTGase) discovered to date; (c) contains at least sixoverlapping copies of epitopes known to be recognized by patient derivedT cells; (d) stimulates representative T cell clones that recognizethese epitopes with sub-micromolar efficacy; and (e) has homologs inproteins from all toxic foodgrains but no homologs in non-toxicfoodgrain proteins. In aggregate, these findings demonstrate that theonset of symptoms upon gluten exposure in the Celiac Sprue patient canbe traced back to a small segment of α-gliadin. Finally, it is shownthat this “super-antigenic” long peptide can be detoxified in vitro andin vivo by treatment with bacterial prolyl endopeptidase, providing apeptidase therapy for Celiac Sprue.

[0099] Identification of Stable Peptides from Gastric Protease,Pancreatic Protease and Brush Border Membrane Peptidase CatalyzedDigestion of Recombinant α2-gliadin:

[0100] The protein α2-gliadin, a representative α-gliadin (Arentz-Hansenet al. (2000) Gut 46:46), was expressed in recombinant form and purifiedfrom E coli. The α2-gliadin gene was cloned in pET28a plasmid (Novagen)and transformed into the expression host BL21 (DE3) (Novagen). Thetransformed cells were grown in 1-liter cultures of LB media containing50 μg/ml of kanamycin at 37° C. until the OD600 0.6-1 was achieved. Theexpression of α2-gliadin protein was induced with the addition of 0.4 mMisopropyl β-D-thiogalactoside (Sigma) and the cultures were furtherincubated at 37° C. for 20 hours. The cells expressing the recombinantα2-gliadin were centrifuged at 3600 rpm for 30 minutes. The pellet wasresuspended in 15 ml of disruption buffer (200 mM sodium phosphate; 200mM NaCl; 2.5 mM DTT; 1.5 mM benzamidine; 2.5 mM EDTA; 2 mg/L pepstatin;2 mg/L leupeptin; 30% v/v glycerol) and lysed by sonication (1 minute;output control set to 6). After centrifugation at 45000 g for 45 min,the supernatant was discarded and the pellet containing gliadin proteinwas resuspended in 50 ml of 7 M urea in 50 mM Tris (pH=8.0). Thesuspension was again centrifuged at 45000 g for 45 min and thesupernatant was harvested for purification. The supernatant containingα2-gliadin was incubated with 1 ml of nickel-nitrilotriacetic acid resin(Ni-NTA; Qiagen) overnight and then batch-loaded on a column with 2 mlof Ni-NTA. The column was washed with 7M urea in 50 mM Tris (pH=8.0),and α2-gliadin was eluted with 200 mM imidazole, 7 M urea in 50 mM Tris(pH=4.5). The fractions containing α2-gliadin were pooled into a finalconcentration of 70% ethanol solution and two volumes of 1.5M NaCl wereadded to precipitate the protein. The solution was incubated at 4° C.overnight and the final precipitate was collected by centrifugation at45000 g for 30 min, rinsed in water, and re-centrifuged to remove theurea. The final purification step of the α-2 gliadin was developed withreverse-phase HPLC. The Ni-NTA purified protein fractions were pooled in7 M urea buffer and injected to a Vydac (Hesperia, Calif.) polystyrenereverse-phase column (i.d. 4.6 mm×25 cm) with the starting solvent (30%of solvent B: 1:1 HPLC-grade acetonitrile/isopropanol:0.1% TFA). SolventA was an aqueous solution with 0.1% TFA. The separation gradientextended from 30-100% of solvent B over 120 min at a flow rate of 0.8ml/min. TABLE 2 Amount of Peptides Digested after 15 hours 33-merControl A Control B H1P0 <20% >90% >90% H2P0 <20% >61% >85% H3P0<20% >87% >95% H4P0 <20% >96% >95% H5P0 <20% >96% >95%

[0101] The purity of the recombinant gliadin was >95%, which allowed forfacile identification and assignment of proteolytic products byLC-MS/MS/UV. Although many previous studies utilized pepsin/trypsintreated gliadins, it was found that, among gastric and pancreaticproteases, chymotrypsin played a major role in the breakdown ofα2-gliadin, resulting in many small peptides from the C-terminal half ofthe protein and a few longer (>8 residues) peptides from the N-terminalhalf, the most noteworthy being a relatively large fragment, the 33-mer(SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (residues 57-89). Thispeptide was of particular interest for two reasons: (a) whereas mostother relatively stable proteolytic fragments were cleaved to smallerfragments when the reaction times were extended, the 33-mer peptideremained intact despite prolonged exposure to proteases; and (b) threedistinct patient-specific T cell epitopes identified previously arepresent in this peptide, namely, (SEQ ID NO:20) PFPQPQLPY, (SEQ IDNO:21) PQPQLPYPQ (3 copies), and (SEQ ID NO:22) PYPQPQLPY (2 copies).

[0102] To establish the physiological relevance of this peptide,composite gastric/pancreatic enzymatic digestion of α2 gliadin was thenexamined. As expected, enzymatic digestion with pepsin (1:100 w/wratio), trypsin (1:100), chymotrypsin (1:100), elastase (1:500) andcarboxypeptidase (1:100) was quite efficient, leaving behind only a fewpeptides longer than 9 residues (the minimum size for a peptide to showclass II MHC mediated antigenicity) (FIG. 4). In addition to theabove-mentioned 33-mer, the peptide (SEQ ID NO:23) WQIPEQSR was alsoidentified, and was used as a control in many of the following studies.The stability of the 33-mer peptide can also be appreciated whencomparing the results of a similar experiment using myoglobin (anothercommon dietary protein). Under similar proteolytic conditions, myoglobinis rapidly broken down into much smaller products. No long intermediateis observed to accumulate.

[0103] The small intestinal brush-border membrane (BBM) enzymes areknown to be vital for breaking down any remaining peptides fromgastric/pancreatic digestion into amino acids, dipeptides or tripeptidesfor nutritional uptake. Therefore a comprehensive analysis of gliadinmetabolism also required investigations into BBM processing of gliadinpeptides of reasonable length derived from gastric and pancreaticprotease treatment. BBM fractions were prepared from rat smallintestinal mucosa. The specific activities of known BBM peptidases wereverified to be within the previously reported range. Whereas thehalf-life of disappearance of WQIPEQSR was ˜60 min. in the presence of12 ng/μl BBM protein, the half-life of (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF digestion was >20 h. Therefore, thelatter peptide remains intact throughout the digestive process in thestomach and upper small intestine, and is poised to act as a potentialantigen for T cell proliferation and intestinal toxicity in geneticallysusceptible individuals.

[0104] Verification of proteolytic resistance of the 33-mer gliadinpeptide with brush border membrane preparations from human intestinalbiopsies: to validate the conclusions reached as described in Example 1,which describes studies with rat BBM preparations, in the context ofhuman intestinal digestion, BBM preparations were prepared from a panelof adult human volunteers, one of whom was a Celiac Sprue patient inremission, while the rest were found to have normal intestinalhistology. (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, (SEQ IDNO:1) QLQPFPQPQLPY (an internal sequence from the 33-mer used as acontrol), WQIPEQSR and other control peptides (100 μM) were incubatedwith BBM prepared from each human biopsy (final aminopeptidase Nactivity of 13 μU/μl) at 37° C. for varying time periods. While (SEQ IDNO:1) QLQPFPQPQLPY, (SEQ ID NO:23) WQIPEQSR and other control peptideswere completely proteolyzed within 1-5 h, the long peptide remainedlargely intact for at 19 hours. These results confirm the equivalencebetween the rat and human BBM for the purpose of this study. Moreover,these results indicate that the methods, foodstuffs, and other reagentsof the invention can be used in humans not known to have Celiac Sprue toimprove digestion and reduce any ill effects of the long peptide.

[0105] Verification of Proteolytic Resistance of the 33-mer GliadinPeptide in Intact Animals:

[0106] The proteolytic resistance of the 33-mer gliadin peptide,observed in vitro using BBM from rats and humans, was confirmed in vivousing a perfusion protocol in intact adult rats (Smithson and Gray(1977) J. Clin. Invest. 60:665). Purified peptide solutions wereperfused through a 15-20 cm segment of jejunum in a sedated rat with aresidence time of 20 min, and the products were collected and subjectedto LC-MS analysis. Whereas >90% of (SEQ ID NO:1) QLQPFPQPQLPY wasproteolyzed in the perfusion experiment, most of the 33-mer gliadinpeptide remained intact. These results demonstrate that the 33-merpeptide is very stable as it is transported through the mammalian uppersmall intestine. The data is shown in FIG. 5.

[0107] The 33-mer gliadin peptide is an excellent substrate for tTGase,and the resulting product is a highly potent activator ofpatient-derived T cells: studies have demonstrated that regiospecificdeamidation of immunogenic gliadin peptides by tTGase increases theiraffinity for HLA-DQ2 as well as the potency with which they activatepatient-derived gluten-specific T cells. It has been shown that thespecificity of tTGase for certain short antigenic peptides derived fromgliadin is higher than its specificity toward its physiological targetsite in fibronectin; for example, the specificity of tTGase for theα-gliadin derived peptide (SEQ ID NO:3) PQPQLPYPQPQLPY is 5-fold higherthan that for its target peptide sequence in fibrinogen, its naturalsubstrate. The kinetics and regiospecificity of deamidation of the33-mer α-gliadin peptide identified as above were therefore measured.The k_(cat)/K_(M) was higher than that reported for any peptide studiedthus far: kcat/KM=440 min−1 mM−1 for (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF compared to kcat/KM=82 min−1 mM−1 forPQPQLPY and kcat/KM=350 min−1 mM−1 for (SEQ ID NO:3) PQPQLPYPQPQLPY.

[0108] Moreover, LC-MS-MS analysis revealed that the peptide (SEQ IDNO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF was selectively deamidated bytTGase at the underlined residues. Because tTGase activity is associatedwith the brush border membrane of intestinal enterocytes, it is likelythat dietary uptake of even small quantities of wheat gluten will leadto the build-up of sufficient quantities of this 33-mer gliadin peptidein the intestinal lumen so as to be recognized and processed by tTGase.

[0109] Structural Characteristics of the 33-mer Gliadin Peptide and ItsNaturally Occurring Homologs:

[0110] Sequence alignment searches using BLASTP in all non-redundantprotein databases revealed several homologs (E-value<0.001) of the33-mer gliadin peptide, shown in FIG. 6. Interestingly, foodgrainderived homologs were only found in gliadins (from wheat), hordeins(from barley) and secalins (from rye), all of which have been proven tobe toxic to Celiac Sprue patients. Nontoxic foodgrain proteins, such asavenins (in oats), rice and maize, do not contain homologous sequencesto the 33-mer gliadin. In contrast, a BLASTP search with the entireα2-gliadin sequence identified foodgrain protein homologs from bothtoxic and nontoxic proteins. Based on available information regardingthe substrate specificities of gastric, pancreatic and BBM proteases andpeptidases, it is believed that, although most gluten homologs to the33-mer gliadin peptide contained multiple proteolytic sites and aretherefore unlikely to be completely stable toward digestion, severalsequences from wheat, rye and barley are expected to be resistant togastric and intestinal proteolysis. The stable peptide homologs to the33-mer α2-gliadin peptide are (SEQ ID NO:24)QPQPFPPQLPYPQTQPFPPQQPYPQPQPQYPQPQ (from α1- and α6-gliadins); (SEQ IDNO:25) QQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPF (from B1 hordein); (SEQ IDNO:26) QPFPQPQQTFPQQPQLPFPQQPQQPFPQPQ (from γ-gliadin); (SEQ ID NO:27)QPFPQPQQPTPIQPQQPFPQRPQQPFPQPQ (from ω-secalin). These stable peptidesare all located at the N-terminal region of the corresponding proteins.The presence of proline residues after otherwise cleavable residues inthese peptides would contribute to their proteolytic stability.

[0111] Bacterial Prolyl Endopeptidase Rapidly Detoxifies the 33-merGliadin Peptide:

[0112] The abundance and location of proline residues is a crucialfactor contributing to the resistance the 33-mer gliadin peptide towardgastrointestinal breakdown. In accordance with the methods of theinvention, a prolyl endopeptidase can catalyze breakdown of thispeptide, thereby diminishing its toxic effects. Preliminary in vitrostudies with short gliadin peptides and the prolyl endopeptidase (PEP)from F. meningosepticum demonstrate this aspect of the invention. Theability of this PEP to clear the 33-mer gliadin peptide was evaluatedvia in vitro and in vivo experiments. Using both rat BBM andco-perfusion of the peptide and PEP in intact rat intestines, thisdetoxification was demonstrated. The results are shown in FIG. 7.Together these results highlight the potential of detoxifying gluten inCeliac Sprue patients by peptidase therapy.

[0113] Although gluten proteins from foodgrains such as wheat, rye andbarley are central components of a nutritious diet, they can beextremely toxic for patients suffering from Celiac Sprue. To elucidatethe structural basis of gluten toxicity in Celiac Sprue, comprehensiveproteolytic analysis was performed on a representative recombinantgliadin under physiologically relevant conditions. An unusually long andproteolytically stable peptide product was discovered, whosephysiological relevance was confirmed by studies involving brush bordermembrane proteins from rat and human intestines as well as intestinalperfusion assays in live rats. In aggregate, these data demonstrate thatthis peptide and its homologs found in other wheat, rye and barleyproteins contribute significantly to the inflammatory response todietary wheat in Celiac Sprue patients.

[0114] The absence of satisfactory animal models for Celiac Sprueimplies that the pivotal pathogenic nature of the gluten peptidesidentified in this study can only be verified in human patients. Whilethis is likely to be a formidable task, and would in any event need tobe conducted in a manner that would not harm the patient, the resultsabove demonstrate that the deleterious effects of gluten ingestion byCeliac Sprue patients can be amelioriated by enzyme treatment of glutencontaining foods. Specifically, co-adminstration of a bioavailable formof a suitable prolyl endopeptidase with dietary gluten would attenuateits toxicity by cleaving the stable 33-mer peptide into non-immunogenicproducts. Given the absence of a satisfactory therapeutic option forCeliac Sprue and the notorious difficulty associated with long-termmaintenance of a gluten-free diet, the peptidase therapies of thepresent invention provides an alternative to strict abstinence for therapidly growing numbers of individuals affected by this disease.

EXAMPLE 3 Peptidase Supplementation as Therapy for CeliacSprue—Demonstration of Efficacy and Safety in Rats and Humans In Vivo

[0115] As described above, Celiac Sprue is a disease engendered by thegliadin peptides in wheat, rye, or barley that interact with the smallintestine to produce a cascade of events leading to the destruction ofintestinal mucosa and consequent malabsorption of nutrients andvitamins. Gliadin peptides are highly resistant to digestion by gastric,and pancreatic proteases as well as by the integral peptidases of theintestinal brush border surface. The interaction of recombinantα-gliadin with mammalian pepsin, chymotrypsin, trypsin, and elastase hasshown that a 33-residue peptide rich in glutamine(Q) and proline(P)residues ((SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) is a majorfinal digestion product. When this 33-mer is exposed to intact rat smallintestine or to human intestinal brush border membranes, it isimpervious to additional breakdown. This peptide has very highspecificity for stimulating T cell proliferation in peripheral bloodlymphocyte cultures from Celiac Sprue patients but not in lymphocytecultures from normal individuals. Because of the resistance of this andother gliadin peptides to pancreatic and intestinal digestion and theabundance of proline residues in these peptides, the 33-mer and othergliadin peptides were exposed to a prolyl endopeptidase, whichdemonstrated that this enzyme is highly effective under physiologicconditions in breaking the peptide bonds between the proline and thenext residue on the peptide chain. The consequent cleavage of thegliadin peptide rendered it incapable of inducing lymphocyteproliferation, demonstrating that the additional processing of thegliadin peptide should prevent its toxic reaction to the intestine inCeliac Sprue patients.

[0116] This example describes experiments to demonstrate that a PEP iseffective in further digesting the toxic gliadin peptides underphysiological conditions. Celiac Sprue patients, in accordance with themethods of the present invention, can consume a normal diet along with aPEP supplement that will digest the toxic gliadin peptide and circumventthe reaction that leads to T-cell proliferation and destruction of theintestinal mucosa. This is an alternative and innovative treatment inCeliac Sprue—the substitution of a rigid gluten-free diet with anexogenous digestive endopeptidase that promotes metabolism and essentialdetoxification of the gliadin peptide. The studies described in thisExample can be used to document efficacy and safety and include a pilotstudy in controls and Celiac patients with PEP-treated wheat flour.These are important studies that enable the institution of a fullclinical trial in normal humans and those with Celiac Sprue.

[0117] The studies described in this example include the following:

[0118] 1. To determine whether exogenous peptidase supplementationdigests resistant gliadin peptides to non-toxic, absorbable products inthe rat in vivo under physiologic conditions. This study involves:

[0119] A. Expression and purification of recombinant ProlylEndopeptidase (rPEP);

[0120] B. Examination of rPEP action on gliadin peptides in ratintestine in vivo; and determination of optimal conditions for efficientdigestion and analysis of effects on intestinal structure and functionwith acute and chronic administration.

[0121] 2. To perform preliminary clinical testing of the efficacy of PEPin processing the resistant gliadin peptides in wheat flour to non-toxicproducts.

[0122] 3. To establish the ideal conditions for packaging the rPEP toachieve efficient digestion of gliadin peptides in vivo, including thepreparation of formulations of rPEP (polyanhyride capsules,methacrylate-glycol capsules; OROS).

[0123] As described in the preceding examples, experiments involvingexposure of α-gliadin to purified pancreatic proteases have demonstratedthe production of a 33-residue glutamine and proline-rich peptide ((SEQID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) as a major end product.When this peptide is administered by perfusion into the small intestineof the intact rat under physiologic conditions or incubated with humanintestinal brush border membranes, its digestion is relatively retardedas compared to that for most dietary peptides such as myoglobin fromperipheral muscle. Experiments demonstrate that a prolyl endopeptidase(PEP) (from Flavobacterium meningosepticum) at molar concentrations onlyone one-hundredth of those of the digestive resistant 33-mer gliadinpeptide is capable of efficiently cleaving it to smaller peptides thatare 1) non-toxic residual peptides (as estimated from the human T cellproliferation assay), and 2) can be readily further digested andabsorbed by the rat intestine. The conditions for optimal action of thePEP on the resistant α-gliadin 33-mer peptide and other gliadin peptidesthat react in the T cell proliferation asay can be determined by themethods set forth in this example.

[0124] To demonstrate that exogenous peptidase supplementation digestsresistant gliadin peptides to non-toxic, absorbable products in vivounder physiologic conditions, expression and purification of recombinantProlyl Endopeptidase (rPEP) can be undertaken. Recombinant prolylendopeptidase (rPEP) from Flavobacterium meningosepticum can beconstructed and expressed as detailed by Yoshimoto Tet et al., and byUchiyama et al. One can also obtain recombinant preparations of a PEPenzyme from Aeromonas Hydrophila as detailed by Shen et al. or fromSphingomonas capsulata (Kabashima T, Fujii M, Meng Y, Ito K, YoshimotoT., Prolyl endopeptidase from Sphingomonas capsulate: isolation andcharacterization of the enzyme and nucleotide sequence of the gene, ArchBiochem Biophys. Oct. 1, 1998; 358(1):141-8.)

[0125] To demonstrate rPEP action on gliadin peptides in rat intestinein vivo and to determine optimal conditions for efficient digestion,intestinal perfusion studies in intact rats can be performed as follows.Sprague-Dawley rats (300-400 gms) are anesthetized with pentobarbital,the abdomen entered through a midline incision, and a 10-20-cm length ofjejunum isolated and catheterized as detailed previously. A test peptide(1 mM GLGG) known to be digested efficiently at the intestinal surfaceis perfused through the isolated segment at 0.4 ml per min in 154 mMNaCl/0.1 % polyethylene glycol to allow calculation for any water flux.The disappearance of the test peptide and the appearance of any productswill allow calculation of the intestinal surface digestion andabsorption. These results are compared with those with the digestiveresistant gliadin peptides (SEQ ID NO:1) QLQPFPQPQLPY, (SEQ ID NO:3)PQPQLPYPQPQLPY, and the 33-mer (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. The GLGG peptide is readilyhydrolyzed to free Leu and Gly and to the dipeptide GG; the high prolinecontent of the gliadin peptides makes them poor substrates for theavailable intestinal membrane peptidases. The intestinal luminal samplestaken at the site of the distal catheter are analyzed by LiquidChromatography-Mass Spectrometry (SpectraSystem, ThermoFinnigan) on aC18 reversed phase column, as previously detailed. Peptide fragments aredetected and their identities confirmed by mass spectrometryfragmentation patterns under conditions where there is a linearrelationship of these peptides and their products.

[0126] After the relative degree of digestion and absorption of the GLGGand gliadin peptides has been established, experiments to demonstratethe efficacy of PEP in digesting the peptides in this in vivo rat modelcan be performed. Initially, the PEP is perfused via a separate catheterat the proximal infusion site of the isolated jejunal segment at molarconcentrations ranging from 1:1000 to 1:1 of that of the test peptides.Preliminary experiments show that a molar ratio of PEP:peptide of 1:100is sufficient for efficient cleavage at the C-terminus of internalProlyl residues to the gliadin sequence. But, it is also important totest the PEP at higher concentrations, in case more peptidase activityis required and desired for total cleavage of the gladin peptides and toassess side effects on the integrity of the intestine and other organs.After each experiment, the intestine and other abdominal organs(especially liver and kidney) are recovered, aliquots quick frozen andpreserved at −70° for subsequent assay of intestinal carbohydrases(sucrase, lactase, maltase) and peptidases (aminopeptidase N,carboxypeptidase, dipeptidyl peptidase IV), and the tissues are fixed informalin, stained for hematoxylin-eosin, and examined (“blindly” withoutknowledge of the experimental protocol) for any histological changes,with particular attention paid to any structural effects that might beproduced of the the higher PEP concentrations.

[0127] Once the ideal ratio of PEP to gliadin peptide is determined inthese perfusion experiments, one can analyze the capacity of the PEP toenhance the hydrolysis of gluten peptides in commercialgluten-containing wheat flour. A 1% slurry of the flour mixed with 1:100(weight basis) trypsin and chymotrypsin, and 1:500 (weight basis)elastase is perfused into the intestine with or without co-perfusion ofsuitable quantities of the PEP. LC-MS analysis of the residual gliadinproducts is conducted on the collected samples, and the histologic andenzymatic parameters are examined, as described above.

[0128] Feeding studies in intact rats can be conducted as follows. Oncethe ideal ratio of the PEP to the gliadin substrate has been establishedin the perfusion experiments, rats are fed 70% carbohydrate chowcontaining wheat flour, which is used as the conventional rat chow forperiods of two weeks. Control rats are fed only the special chow, andthe treated rats are given sufficient PEP supplementation (molar ratiosPEP to gliadin protein: 1:1, 1:10 and 1:100) in the diet to digest theresidual gliadin peptides such as the Pro- and Gln-rich 33-mer. Aftertwo weeks of ab lib feeding, the rats' daily consumption of food isquantified by daily weighing of the residual chow in the feeder and thenutritional assessment determined by daily body weights. Over thefeeding period of 2-4 weeks, rats are weighed and examined daily toverify normal activities and are then killed by stunning anddecapitation. The intestine, liver and kidneys are recovered andexamined for gross and histological integrity, and any anatomicdifferences are noted between the control (PEP−) and treated (PEP+)animals. In addition, digestive enzymes (carbohydrases and proteases)are determined, as detailed for the rat perfusion studies.

[0129] Preliminary clinical testing of the efficacy of PEP in processingthe resistant gliadin peptides can be conducted as follows. Now that ithas been established that PEP can readily convert the high-Pro, high-Glngliadin peptides to smaller, non-toxic fragments that do not produceproliferation in the T cell assay, preliminary testing of PEP treatedwheat flour containing the usual or enhanced amounts of gluten (e.g.,Bob's RedMill flour, Milwaukee, Oreg.) or food-grade gluten or gliadinitself (e.g. gliadin from Sigma Aldrich) can be undertaken. The flour,gluten or gliadin can be batch treated with appropriate amounts ofpurified mixtures of pancreatic enzymes that are used clinically totreat pancreatic insufficiency (e.g., Pancrease MT 20 containing 20,000units lipase, 44000 units of the pancreatic proteases and 56000 units ofamylase per capsule). Incubation of a slurry of flour, gluten or gliadinwith the material from an appropriate amount of capsules of thePancrease preparation can be carried out in 0.02 M Na—K phosphatebuffer, pH 6.5 at 37° C. for several hours under sterile conditionsuntil 1) standard T cell proliferation assays (see, for example,Arentz-Hansen, 2000) identifies the highly active signal produced by thegliadin peptides and particularly 33-mer, ((SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) and 2) the average size of theproteolyticaly derived gliadin peptides has been reduced to <6 residues(as measured by gel filtration HPLC). The pre-digested flour, gluten orgliadin is then exposed to sufficient pure PEP (for example, 1 molePEP:100 moles gliadin substrate) under sterile conditons for 1-18 hours,and the cleavage of gliadin peptides with known toxicity such as the33-mer verified by LC-MS analysis. In parallel control studies,previously denatured PEP (by heating to 90° C. for 60 min) can beincubated with the protease-treated flour and the persistence of thesetoxic peptides is verified by LC-MS analysis. These tests demonstratethe usefulness of a the 33-mer in assays; in one aspect, the presentinvention provides the 33-mer in isolated and purified forms, as well asassays to detect its present in foodstuffs.

[0130] The pre-treated flours can be incorporated into otherwisegluten-free breakfast muffins by a nutritionist, and these served tovolunteer persons and those with biopsy-proven Celiac Sprue at a“community” breakfast in the nutrition department for a period of twoweeks. Patients must have uncomplicated Celiac Sprue that is inremission on gluten exclusion alone. Control volunteers who have beenestablished not to have Celiac Sprue and negative Celiac antibodystudies are also recruited. During this period the control muffins madewith flour that has been treated with denatured pancreatic proteases±PEPare given. The PEP+ muffins are given for the first two weeks followedby a two week break from the breakfasts, and the PEP− muffins areadministered over the second two week breakfast sessions. The study canbe single-blinded, the subjects being unaware of whether PEP is includedin the study. The physician and nutritionist will know the flour hasbeen exposed only to the pancreatic proteases or also to PEP, in casethere are any untoward reactions to the PEP material. All study subjectswill fill out a questionnaire regarding their observations during eachtwo week period as well as during the two week break time and the twoweeks after the second muffin breakfast period. Although obtaining abiopsy via endoscopy would be an ideal monitor of the PEP efficacy, thiscannot be ethically justified based on currently available data.Endoscopy may be offered only if needed as an aspect of patient care.Participants will initially meet briefly with the responsiblephysician-investigator who will be available throughout the study.Participants will be interviewed and the questionnaire reviewed by anutritionist and physician before the study, at the end of each two weekperiod and two weeks after completing the study. The principalinvestigator will be ultimately responsible for the conduct of the trialand will meet regularly with the responsible physician and nutritionistto whom the day to day aspects of the study will be delegated. Adultsfrom age 17 and older can be eligible for the study. Both males andfemales with Celiac Sprue will be recruited through Celiac supportorganizations. Individuals from various ethnic groups, including Asianand African American can be recruited, although most patients withCeliac Sprue are Caucasians. Both males and females can participate;there is a somewhat higher proportion of female Celiac Sprue patients(˜65%). Participants will have 24 hour access to the gastroenterologyteam, and a member of the research team will be available forconsultation. Efficacy will be monitored by the comparative responses ofparticipants during the control period when ingesting protease-treatedflour without the PEP versus the same flour that has been treated withPEP.

[0131] Suitable conditions for packaging the rPEP to achieve efficientdigestion of gliadin peptides in vivo can be determined as follows. Todevelop a palatable preparation of PEP to enable the in vivo digestionof the toxic peptides in humans, it can be useful to formulate PEP sothat it can pass into the small intestine without being destroyed by theharsh acidic environment of the stomach. In addition, this formulationcan provide rapid release of PEP upon entry into the duodenum, where thesecreted pancreatic proteases exert their maximal action within theluminal contents to cleave dietary proteins. There are severalwell-studied and widely used examples of such delivery systems for othersubstances. The development of an optimized formulation for an effectivePEP drug capable of delivering pharmacologically useful quantities ofthis enzyme into the upper small intestine as a digestive supplement canbe conducted as follows. To process the digestive-resistant gliadinpeptides, selected formulation strategies that have been usedsuccessfully for the delivery of other enzyme supplements can be used.In particular, previously used formulations for pancreatic proteases andlactase are evaluated by use of recombinant PEP from Flavobacteriummeningoscepticum and Aeromonas hydrophila. These enzymes are expressedand purified as described by A. Kitazono et al. and A. Kanatani et al.Pancreatic enzymes have been used for the past seventy years to treatpancreatic exocrine insufficiency. Although early clinical results werevariable due to gastric inactivation of the exogenously administeredenzymes, a revived interest in enzyme-containing digestive aids occurredaround 1960 with the development of acid stable enteric coatings (I. R.Wilding, S. S. Davis, and D. T. O'Hagan, Targeting of drugs and vaccinesto the gut. Pharmac. Ther. 62, 97-124, (1994)). Similarly, acid stableenteric coatings have also been used for the delivery of lactase intothe duodenum of patients with lactase deficiency. In one embodiment, theglutenase formulations of the invention comprise a glutenase in a stableenteric coating.

[0132] Lyophilized, particulate PEP mixed with bicarbonate (as buffer)is coated with Eudragit S100, L30D or L 100-44 according tomanufacturer's instructions (Rohm America). Alternatively, celluloseacetate phthalate, methylcellulose or hydroxypropylmethyl cellulosephthalate can be used as coatings for the preparation of gastric acidresistant pellets. These enteric coatings are commonly used for theformulation of pancreatin (see T. Sipos (1978), Preparation of entericcoated digestive enzyme compositions, U.S. Pat. No. 4,079,125; and T.Sipos (1998), High buffer-containing enteric coating digestive enzymebile acid compositions and method of treating digestive disorderstherewith, U.S. Pat. No. 5,750,104).

[0133] An alternative strategy useful in preparing formulations of theinvention, used successfully with lactase (B. J. Langner (1999), Entericpolymer coated capsule containing dried bacterial culture for supplyinglactase, U.S. Pat. No. 6,008,027), involves filling gelatin capsuleswith 50-90% lyophilized PEP, the remaining capacity being filled withstabilizing dessicants such as silicon oxide, silicon dioxide ormicrocrystalline cellulose and bicarbonate buffer. The capsules areenterically coated with Eudragit polymer (Rohm America) or polyvinylacetate phthalate (Sureteric, Merck Frosst) and vacuum dried prior touse. Similarly, diastase has been formulated with Eudragits RS100 andcellulase acetate phthalate coatings for enteric use (S. P. Vyas, P. J.Gogoi, S. Pande, and V. K. Dixit, Enteric spherules diastase in enzymepreparations. J. Microencapsulation. 8, 447-454, 1991). To demonstratethat these or other formulations increase PEP bioavailability in thesmall intestine, one can perform the following experiments. First, theability of PEP activity to withstand 0.5-2 h of simulated gastrictreatment (pepsin, in 0.1N HCl, pH 2) can be evaluated. If >10% activitycan be reproducibly retained, the formulation is exposed to simulatedconditions in the duodenum (pH 6.5 buffer containing trypsin,chymotrypsin and carboxypeptidase at a 1:100 molar ratio and elastase ata 1:500 ratio to the putative α2-gliadin). Ideally, full release of PEPactivity would be achieved within 15 minutes. Formulations that satisfythe above criteria are fed initially to adult rats in conjunction withgluten-free meals spiked with recombinant α2-gliadin (whose proteolyticbehavior in response to gastric and pancreatic enzymes+PEP has been wellcharacterized). PEP doses in the range of 10-1000 units/kg body weightcan be evaluated. Animals are sacrificed two hours after meals, and thesmall intestinal derived contents are analyzed by LC-MS for residual PEPactivity and the extent to which gliadin has been proteolyzed. Inparticular, the concentration of the 33-mer digestive-resistant gliadinpeptide is estimated. Formulations that yield >90% reduction inconcentration of this peptide are evaluated more extensively forpotential toxicity, as detailed above for the initial rat studies withwater soluble PEP.

[0134] The procedures described herein are performed under an approvedAnimal Protocol described below. Male Sprague-Dawley rats, 250-300 g,(or Fisher rats for studies of DPP IV deficient intestine) are allowedaccess to regular wheat-based rat chow until the experiment. Rats areallowed water only for 8 hours prior to the experiment to insureclearance of residual chow in the upper small intestine. After the ratis anesthetized with an intraperitoneal injection of pentobarbital (50mg/Kg), the abdominal cavity is opened and a small incision made in asegment of jejunum located 10 cm beyond the ligament of Trietz.Cannulation is made with a polyethylene catheter (3 mm ID, 4 mm OD) andsutured 2 cm distal to the incision. A second cannula is placed insimilar fashion 10 cm distal to the first with the cannula facingproximally. After rinsing the isolated, intact jejunal segment withRinger's solution (140 mM NaCl, 10 mM KHCO₃, 1.2 mM K₂HPO₄, 1.2 mMCaCl₂, 1.2 mM MgCl₂) at 37° C. to remove any intraluminal debris, theisolated loop of intestine is returned to the abdominal cavity. Theincision is covered with clear plastic wrap, and intra-abdominaltemperature maintained at 37° C. by positioning a 30 watt incandescentlap at ˜30 cm from the animal. A 2 mM solution of a gliadin peptide of7-14 residues (purified and characterized by HPLC-Mass Spectrometry) isperfused in Ringer's solution containing [¹⁴C]inulin ( adilution-concentration marker) to establish a steady state ofconcentrations of residual peptide and smaller products at thecollection distal collection site (previous studies with other peptidesand carbohydrates have revealed the steady state to be achieved in 10-20minutes). Samples collected at the distal site are recovered andanalyzed by HPLC-MS for residual peptide and smaller peptide or aminoacid products. Samples are collected over 3 successive 10 minute periodsafter a steady state is achieved, and a series of gliadin andnon-gliadin peptides are used. Animals can usually be maintained underanesthesia for a period of 3 to 6 hours by the addition of smallincrements of pentobarbital (˜5 mg per 30-60 minutes). At the end of theexperiment, the intestinal segment and an adjacent control segment arerecovered and samples taken from liver, kidney and blood for analysis ofthe test peptide and its products. Terminal euthanasia is accomplishedby an overdose of anesthesia to produce apnea until there is no heartcontraction.

[0135] While other methods and reagents can be employed for purposes ofthe present invention, this example provides enzymes, enzymeformulations, and animal and clinical testing protocols to demonstratethe efficacy of enzyme-mediated therapy for Celiac Sprue.

EXAMPLE 4 Heterologous Expression of PEP in Lactobacilli

[0136] In one embodiment of the present invention, a Celiac Spruepatient is provided with a recombinant organism modified to express aPEP of the invention. The recombinant organism is selected from thoseorganisms that can colonize the intestinal mucosa without detriment tothe patient, thereby providing an endogenous source of PEP to thepatient. As one example, Lactobacilli such as L. casei and L. plantariumcan colonize the intestinal mucosa and secrete PEP enzymes locally.Given their widespread use in food processing, they can also be used asan efficient source of PEP for industrial (to treat foodstuffs) andmedical (to prepare PEP for pharmaceutical formulation) use. PEPs can beexpressed in such lactobacilli using standard recombinant DNAtechnologies. For example, Shaw et al. (Shaw, D M, Gaerthe, B; Leer, RJ, Van der Stap, J G M M, Smittenaar, C.; Den Bak-Glashouwer, Heijne, MJ, Thole, J E R, Tielen F J, Pouwels, P H, Havenith, C E G (2000)Immunology 100, 510-518) have engineered Lactobacilli species to expressintracellular and surface-bound tetanus toxin. The intact PEP genes(including leader sequences for efficient bacterial secretion) can becloned into shuttle expression vectors such as pLP401 or pLP503 undercontrol of the (regulatable) amylase promoter or (constitutive) lactatedehydrogenase promoter, respectively. Alternatively, recombinant foodgrade Lactobacilli strains can be generated by site specificrecombination technology (e.g. see. Martin M C, Alonso, J C, Suarez J E,and Alvarez M A Appl. Env. Microbiol. 66, 2599-2604, 2000). Standardcultivation conditions are used for Lactobacilli fermentation, such asthose described by Martin et al.

EXAMPLE 5 Heterologous Expression of PEP in Yeasts

[0137] Both naturally occurring and recombinant cells and organisms canbe used to produce the glutenases useful in practice of the presentinvention. Preferred glutenases and producing cells include those fromorganisms known to be Generally Regarded as Safe, such asFlavobacterium, Aeromonas, Sphingomonas, Lactobacillus, Aspergillus,Xanthomonas, Pyrococcus, Bacillus and Streptomyces. Extracellularglutenase enzymes may be obtained from microorganisms such asAspergillus oryzae and Lactobacillus casei. Preferred cells includethose that are already used in the preparation of foodstuffs but havebeen modified to express a glutenase useful in the practice of thepresent invention. As one example, yeast strains such as Saccharomycescerevisiae are useful for high level expression of secreted heterologousproteins. Genes encoding any of the PEPs described above (mature proteinonly) can be cloned in expression plasmids designed for optimalproduction of secreted proteins. An example of such a heterologousexpression strategy is described in Parekh, R. N. and Wittrup, K. D.(Biotechnol. Prog. 13, 117-122, 1997). Either self-replicating (e.g. 2micron) or integrating (e.g. pAUR101) vectors can be used. The GAL1-10promoter is an example of an inducible promoter, whereas the ADH2promoter is an example of a constitutive promoter. The cDNA encoding themature PEP is fused downstream of a leader sequence containing asynthetic pre-pro region that includes a signal cleavage site and aKex2p cleavage site. S. cerevisiae BJ5464 can be used as a host forproduction of the peptidase. Shake-flask fermentation conditions aredescribed by Parekh and Wittrup in the above-cited reference.Alternatively, high cell density fed-batch cultures can be used forlarge scale production of the peptidases; a representative procedure forthis purpose is described in Calado, C. R. C, Mannesse, M., Egmond, M.,Cabral, J. M. S. and Fonseca, L. P. (Biotechnol. Bioeng. 78, 692-698,2002).

EXAMPLE 6 Enteric Capsule Formulation of Prolyl Endopeptidase

[0138] Gelatin capsules are filled with 100 mg prolyl endopeptidase and10 mg of silicon dioxide. The capsules are enterically coated withEudragit polymer and put in a vacuum chamber for 72 hours. The capsulesare then held at a range of temperature of 10° C. to 37° C. and acontrolled humidity level of 35-40%.

EXAMPLE 7 Studies of Enteric Capsule Formulation of Prolyl Endopeptidase

[0139] A study is conducted where patients with Celiac Sprue areenrolled in a two week-long study. Gelatin capsules containing 90%prolyl endopeptidase mixed with 10% silicon dioxide are used. Thecapsules are hand-filled with the mixture, banded, and coated with a 10%Sureteric enteric coating (a polymer of polyvinylacetatephthalatedeveloped by the Canadian subsidiary of Merck & Company). Samples areacid-tested by exposing the coating to 1 N HCL for one hour in order tosimulate the acid environment of the stomach. The capsules are then putin a vacuum chamber for 72 hours.

[0140] Two 100 mg capsules are administered to each patient prior toeach meal. The patients are instructed to eat all kinds of food withoutabstaining from those that were known to cause distress, e.g., bloating,diarrhea, and cramps.

EXAMPLE 8 Enteric Pill Formulation of Prolyl Endopeptidase

[0141] 400 mg of L-tartaric acid and 40 mg of polyethyleneglycol-hydrogenated castor oil (HCO-60) are dissolved in 5 ml ofmethanol. This solution is placed in a mortar previously warmed to 30°C. To the solution is added 100 mg of prolyl endopeptidase. Immediatelyafter the addition of PEP, the mixture is stirred with a pestle under ahot air current (40° C.) and then placed in a desiccator under vacuumovernight to remove the solvent. The resulting solid-mass is pulverizedwith a pestle and kneaded with 30 mg of sodium bicarbonate and a smallamount of 70% ethanol. The mixture is then divided and shaped into pillsof about 2 mm size and thoroughly dried. The dried pills are given acoating of hydroxypropylmethylcellulose phthalate (HP-55) to obtain anenteric formulaton.

EXAMPLE 9 Diagnostic Methods

[0142] The 33-mer peptide ((SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) and its deamidated derivatives that are formed by the action oftissue transglutaminase are useful diagnostic reagents for the detectionof Celiac Sprue. The enzyme tTGase deamidates the 33-mer at least at theunderlined positions shown in the following sequence: (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, and deamidated counterparts of the33-mer are important reagents of the present invention. Such deamidatedcounterparts may comprise one, two or more deamidated glutamine (Q)residues.

[0143] Oligoeptide analogs of the oligopeptides described by amino acidsequence herein are also included. Such analogs contain at least onedifference in amino acid sequence between the analog and nativeantigenic peptide. An L-amino acid from the native peptide may bealtered to any other one of the 20 L-amino acids commonly found inproteins, any one of the corresponding D-amino acids, rare amino acids,such as 4-hydroxyproline, and hydroxylysine, or a non-protein aminoacid, such as β-alanine and homoserine. Also included with the scope ofthe present invention are amino acids that have been altered by chemicalmeans such as methylation (e.g., α-methylvaline), deamidation, amidationof the C-terminal amino acid by an alkylamine such as ethylamine,ethanolamine, and ethylene diamine, and acylation or methylation of anamino acid side chain function (e.g., acylation of the epsilon aminogroup of lysine), deimination of arginine to citrulline,isoaspartylation, or phosphorylation on serine, threonine, tyrosine orhistidine residues. Candidate oligopeptide analogs may be screened forutility in a diagnostic method of the invention by an assay measuringcompetitive binding to antibodies, T cell receptor, etc. Those analogsthat inhibit binding of the native peptides are useful diagnosticreagents. Oligopeptides and oligopeptide analogs may be synthesized bystandard chemistry techniques, including synthesis by automatedprocedure.

[0144] Monoclonal antibodies are provided by the invention, which reactspecifically with this peptide and its deamidated derivatives. Methodsof producing antibodies are wll known in the art. Polyclonal antibodiesare raised by a standard protocol, for example by injecting a productionanimal with an antigenic composition, formulated as described above.See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. When a peptide immunogen is utilized, it isadvantageous to conjugate the peptide with a larger molecule to make animmunostimulatory conjugate. Commonly utilized conjugate proteins thatare commercially available for such use include bovine serum albumin(BSA) and keyhole limpet hemocyanin (KLH). Polyclonal antibodiesspecific for the polypeptide may then be purified from such antisera by,for example, affinity chromatography using the polypeptide coupled to asuitable solid support. Alternatively, for monoclonal antibodies,hybridomas may be formed by isolating the stimulated immune cells, suchas those from the spleen of the inoculated animal. These cells are thenfused to immortalized cells, such as myeloma cells or transformed cells,which are capable of replicating indefinitely in cell culture, therebyproducing an immortal, immunoglobulin-secreting cell line. Many suchcell lines (such as myelomas) are known to those skilled in the art. Inaddition, antibodies or antigen binding fragments may be produced bygenetic engineering. In this technique, as with the standard hybridomaprocedure, antibody-producing cells are sensitized to the desiredantigen or immunogen. The messenger RNA isolated from the immune spleencells or hybridomas is used as a template to make cDNA using PCRamplification. A library of vectors, each containing one heavy chaingene and one light chain gene retaining the initial antigen specificity,is produced by insertion of appropriate sections of the amplifiedimmunoglobulin cDNA into the expression vectors. A combinatorial libraryis constructed by combining the heavy chain gene library with the lightchain gene library. This results in a library of clones which co-expressa heavy and light chain (resembling the Fab fragment or antigen bindingfragment of an antibody molecule). The vectors that carry these genesare co-transfected into a host (e.g. bacteria, insect cells, mammaliancells, or other suitable protein production host cell.). When antibodygene synthesis is induced in the transfected host, the heavy and lightchain proteins self-assemble to produce active antibodies that can bedetected by screening with the antigen or immunogen.

[0145] The 33 mer peptide is exceptionally resistant towardgastrointestinal proteolysis, thereby allowing the peptide to persist asit travels through the intestinal tract. Also, this peptide includesmultiple copies of immunogenic epitopes from gliadin that are recognizedby antibodies in most Celiac patients. Because multivalent epitopes areknown to elicit an especially vigorous immune response (e.g. Boniface etal., 1998, Immunity 9: 459), the 33-mer and its deamidated derivativeshave inflammatory properties in the Celiac intestine, even at low doses.Moreover, as tTGase is known to become transiently linked to itssubstrate, the present invention provides fusion proteins in which allor a portion of a mammalian tTGase, including but not limited to human,bovine, equine, and porcine tTGase, is linked, usually covalently, tothe 33-mer of the invention, wherein the linkage site is at a site foreventual deamidation. This fusion protein of the invention is a highlypotent stimulator of T cells from Celiac Sprue patients in that thefusion protein exactly mimics the complexes formed in Celiac Spruepatients and is recognized by the anti-tTGase antibodies and by T cellsin those patients.

[0146] In one embodiment, the present invention provides a diagnosticfor Celiac Sprue that is a urine test. It is well known that thepermeability of the small intestine increases during active Celiac Sprueand reduces again when a strict gluten-free diet is followed (e.g.Johnston et al., 2001, Lancet 358: 259). As the 33-mer peptide traversesthe small intestine, a small amount of the peptide derived from a testmeal will induce leakiness, and in turn be transported across theepithelial layer, and passed into urine. Given its proteolyticresistance, this peptide will emerge in the urine, and can be detectedby standard analytical procedures such as LC-tandem mass spectrometry oran antibody-based diagnostic test. Presence of the peptide in the urineis diagnostic for Celiac Sprue. The sensitivity of this diagnosticprocedure could be increased through the use of ¹³C or other labeledpeptide. Moreover, in current practice, an individual suspected ofhaving Celiac Sprue is typically placed on a gluten-free diet and thenchallenged with gluten some weeks later to see if symptoms reappear. Thediagnostic tests of the present invention can be used upon thephysician's first suspicion that an individual is suffering from CeliacSprue, thereby avoiding the harmful effects of placing that individualback on a gluten-containing diet and re-inducing the disease symptoms.

[0147] In one embodiment, the present invention provides a diagnosticfor Celiac Sprue that is a blood test. As discussed above, the 33-mercan also be detected in peripheral blood samples, when ingested in smallquantities by Celiac Sprue individuals or at the time of an initialscreening at a physician's office.

[0148] In one embodiment, the present invention provides a diagnosticfor Celiac Sprue that is based on intestinal biopsy staining. Labeledforms of the 33-mer provided by the present invention (e.g. peptideconjugated to a fluorescent or other label) can be used to stainintestinal biopsy samples from Celiac Sprue patients. Due to theirmultivalency and anticipated high affinity for antigen presenting cellsand, in turn, inflammatory T cells, such peptides can be used to detectthe presence of disease specific immune cells in biopsy tissue. Ofparticular relevance is the use of such assays to identify patientswhose disease is in remission as a result of a gluten-free diet. Asnoted above, current clinical practices are unable to diagnose a patientwhen he or she is on a gluten-free diet, and require that the patient besubjected to the discomfort of a gluten containing diet for asignificant time period.

[0149] In one embodiment, the present invention provides a diagnosticfor Celiac Sprue in which labeled forms of the 33-mer are used to detectdisease specific immune cells in peripheral blood.

[0150] In one embodiment, the present invention provides a diagnosticfor Celiac Sprue that is based on an oral mucosa challenge. Inflammatorypeptides from gluten can be used to detect Celiac Sprue by localchallenge on oral mucosa of patients (see Lahteenoja et al., 2000, Am.J. Gastroenterol. 95: 2880). Given the proteolytic resistance andimmunogenicity of the 33-mer, the 33-mer can be especially useful in adiagnostic procedure in which the peptide is contacted with the oralmucosa of an individual, and a diagnosis of Celiac Sprue is made ifinflammation results. Again, a particular advantage of such a test wouldbe its sensitivity to detect a patient whose disease is in remission dueto a gluten-free diet.

[0151] In one embodiment, the diagnosis involves detecting the presenceof T cells reactive with the 33-mer or a deamidated counterpart thereof,or a tTGase-linked counterpart thereof in a tissue, bodily fluid, orstool of an individual. T cells can also be detected by proliferation inresponse to exposure to an antigen provided by the present invention andpresented by autologous or suitable allogeneic antigen presenting cells.The presence of such reactive T cells indicates the presence of anon-going immune response. The antigen used in the assays may be thecomplete 33-mer, deamidated counterpart, or a tTGase-linked counterpart;or peptides derived therefrom, usually such peptides will be at leastabout 12 amino acids in length. A subset of peptides may be prepared, ora mixture that encompasses the complete sequence. Overlapping peptidesmay be generated, where each peptide is frameshifted from 1 to 5 aminoacids, thereby generating a set of epitopes.

[0152] Quantitation of T cells can be performed by determing cognatebinding of the T cell receptor present on a cell, to an MHC/peptidecomplex, e.g. using Class I or Class II MHC tetramers (see Altman et al.Science (1996) 274: 94-96; McMichael and O'Callaghan J Exp Med. (1998)187: 1367-1371). MHC Tetramers are complexes of the soluble fragments offour MHC molecules, which are associated with a specific peptide. Thetetramer may be bound to a fluorochromes or other detectable label. (seeOgg et al. (1998) Curr Opin Immunol. 10: 393-396). The tetramer maycomprise a soluble fragment of HLA-DQ2 [DQ(a1*0501, b1*02)] and/or DQ8[DQ(a1*0301, b1*0302)] molecule, or other MHC types appropriate for theindividual being tested.

[0153] The diagnosis may determine the level of reactivity, e.g. basedon the number of reactive T cells found in a sample, as compared to anegative control from a naive host, or standardized to a data curveobtained from one or more positive controls. In addition to detectingthe qualitative and quantitative presence of antigen reactive T cells,the T cells may be typed as to the expression of cytokines known toincrease or suppress inflammatory responses. While not necessary fordiagnostic purposes, it may also be desirable to type the epitopicspecificity of the reactive T cells, particularly for use in therapeuticadministration of peptides.

[0154] In another embodiment, the diagnosis involves detecting thepresence of an antibody, reactive with the 33-mer or a deamidatedcounterpart thereof, or a tTGase-linked counterpart thereof in a tissue,bodily fluid, or stool of an individual. An antibody is detected by, forexample, an agglutination assay using an antigen provided by the presentinvention. Samples may be obtained from patient tissue, which may be amucosal tissue, including but not limited to oral, nasal, lung, andintestinal mucosal tissue, a bodily fluid, e.g. blood, sputum, urine,phlegm, lymph, and tears. Also included in the term are derivatives andfractions of such fluids. Blood samples and derivatives thereof are ofparticular interest. One advantage of the present invention is that theantigens provided are such potent antigens that the diagnostic methodsof the invention can be employed with samples (tissue, bodily fluid, orstool) in which a Celiac Sprue diagnostic antibody, peptide, or T cellis present in very low abundance. This allows the methods of theinvention to be practiced in ways that are much less invasive, much lessexpensive, and much less harmful to the Celiac Sprue individual.

[0155] Measuring the concentration of specific antibodies in a sample orfraction thereof may be accomplished by a variety of specific assays, asare known in the art. In general, the assay will measure the reactivitybetween a patient sample, usually blood derived, generally in the formof plasma or serum. The patient sample may be used directly, or dilutedas appropriate, usually about 1:10 and usually not more than about1:10,000. Immunoassays may be performed in any physiological buffer,e.g. PBS, normal saline, HBSS, dPBS, etc.

[0156] In one embodiment, a conventional sandwich type assay is used. Asandwich assay is performed by first attaching the peptide to aninsoluble surface or support. The peptide may be bound to the surface byany convenient means, depending upon the nature of the surface, eitherdirectly or through specific antibodies. The particular manner ofbinding is not crucial so long as it is compatible with the reagents andoverall methods of the invention. They may be bound to the platescovalently or non-covalently, preferably non-covalently.

[0157] In some cases, a competitive assay will be used. In addition tothe patient sample, a competitor to the antibodies is added to thereaction mix. The competitor and the antibodies compete for binding tothe antigenic peptide. Usually, the competitor molecule will be labeledand detected as previously described, where the amount of competitorbinding will be proportional to the amount of antibodies present. Theconcentration of competitor molecule will be from about 10 times themaximum anticipated antibodies concentration to about equalconcentration in order to make the most sensitive and linear range ofdetection.

[0158] An alternative protocol is to provide anti-patient antibodiesbound to the insoluble surface. After adding the sample and washing awaynon-specifically bound proteins, one or a combination of the testantigens are added, where the antigens are labeled, so as not tointerfere with binding to the antibodies. Conveniently, fused proteinsmay be employed, where the peptide sequence is fused to an enzymesequence, e.g. β-galactosidase.

[0159] The subject methods are useful not only for diagnosing CeliacSprue individuals but also for determining the efficacy of prophylacticor therapeutic methods for Celiac Sprue as well as the efficacy of foodpreparation or treatment methods aimed at removing glutens or similarsubstances from food sources. Thus, a Celiac Sprue individualefficaciously treated with a prophylactic or therapeutic drug or othertherapy for Celiac Sprue tests more like a non-Celiac Sprue individualwith the methods of the invention. Likewise, the antibodies or T cellresponders, e.g. T cell lines, of the invention that detect the toxicgluten oligopeptides of the invention are useful in detecting gluten andgluten-like substances in food and so can be used to determine whether afood treated to remove such substances has been efficaciously treated.

[0160] These and other diagnostic methods of the invention can bepracticed using the novel peptides and antibodies provided by theinvention.

[0161] All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication, patent, or patent application were specifically andindividually indicated to be incorporated by reference.

[0162] The present invention has been described in terms of particularembodiments found or proposed by the inventor to comprise preferredmodes for the practice of the invention. It will be appreciated by thoseof skill in the art that, in light of the present disclosure, numerousmodifications and changes can be made in the particular embodimentsexemplified without departing from the intended scope of the invention.Moreover, due to biological functional equivalency considerations,changes can be made in methods, structures, and compounds withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

1 27 1 12 PRT Triticum aestivum 1 Gln Leu Gln Pro Phe Pro Gln Pro GlnLeu Pro Tyr 1 5 10 2 12 PRT Triticum aestivum PYRROLIDONE CAR (1)...(1)N terminal pyroglutaminate 2 Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu ProTyr 1 5 10 3 14 PRT Triticum aestivum 3 Pro Gln Pro Gln Leu Pro Tyr ProGln Pro Gln Leu Pro Tyr 1 5 10 4 13 PRT Triticum aestivum 4 Pro Gln ProGln Leu Pro Tyr Pro Gln Pro Gln Leu Pro 1 5 10 5 11 PRT Triticumaestivum 5 Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu Pro 1 5 10 6 11 PRTTriticum aestivum 6 Gln Pro Gln Phe Pro Gln Pro Gln Leu Pro Tyr 1 5 10 79 PRT Triticum aestivum 7 Gln Pro Phe Pro Gln Pro Gln Leu Pro 1 5 8 6PRT Triticum aestivum 8 Pro Gln Pro Gln Leu Pro 1 5 9 13 PRT Triticumaestivum 9 Arg Arg Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg Gly 1 5 10 1010 PRT Triticum aestivum 10 Gln Pro Phe Pro Gln Pro Gln Leu Pro Tyr 1 510 11 7 PRT Triticum aestivum 11 Phe Pro Gln Pro Gln Leu Pro 1 5 12 33PRT triticum aestivum 12 Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu ProTyr Pro Gln Pro 1 5 10 15 Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro TyrPro Gln Pro Gln Pro 20 25 30 Phe 13 10 PRT Triticum aestivum 13 Gln ProGln Gln Ser Phe Pro Gln Gln Gln 1 5 10 14 12 PRT triticum aestivum 14Gln Leu Gln Pro Phe Pro Gln Pro Glu Leu Pro Tyr 1 5 10 15 14 PRTTriticum aestrivum 15 Pro Gln Pro Glu Leu Pro Tyr Pro Gln Pro Glu LeuPro Tyr 1 5 10 16 10 PRT Triticum aestivum 16 Gln Pro Gln Gln Ser PhePro Glu Gln Gln 1 5 10 17 13 PRT Triticum aestivum 17 Pro Gln Pro GluLeu Pro Tyr Pro Gln Pro Gln Leu Pro 1 5 10 18 14 PRT Triticum aestivum18 Pro Gln Pro Glu Leu Pro Tyr Pro Gln Pro Gln Pro Leu Pro 1 5 10 19 5PRT Artificial Sequence Peptide motif 19 Gly Pro Leu Gly Pro 1 5 20 9PRT Artificial Sequence T cell epitope 20 Pro Phe Pro Gln Pro Gln LeuPro Tyr 1 5 21 9 PRT Artificial Sequence T cell epitope 21 Pro Gln ProGln Leu Pro Tyr Pro Gln 1 5 22 9 PRT Artificial Sequence T cell epitope22 Pro Tyr Pro Gln Pro Gln Leu Pro Tyr 1 5 23 8 PRT Artificial SequenceDigestion product 23 Trp Gln Ile Pro Glu Gln Ser Arg 1 5 24 34 PRTTriticum aestivum 24 Gln Pro Gln Pro Phe Pro Pro Gln Leu Pro Tyr Pro GlnThr Gln Pro 1 5 10 15 Phe Pro Pro Gln Gln Pro Tyr Pro Gln Pro Gln ProGln Tyr Pro Gln 20 25 30 Pro Gln 25 35 PRT Triticum aestivum 25 Gln GlnGln Pro Phe Pro Gln Gln Pro Ile Pro Gln Gln Pro Gln Pro 1 5 10 15 TyrPro Gln Gln Pro Gln Pro Tyr Pro Gln Gln Pro Phe Pro Pro Gln 20 25 30 GlnPro Phe 35 26 30 PRT Triticum aestivum 26 Gln Pro Phe Pro Gln Pro GlnGln Thr Phe Pro Gln Gln Pro Gln Leu 1 5 10 15 Pro Phe Pro Gln Gln ProGln Gln Pro Phe Pro Gln Pro Gln 20 25 30 27 30 PRT Triticum aestivum 27Gln Pro Phe Pro Gln Pro Gln Gln Pro Thr Pro Ile Gln Pro Gln Gln 1 5 1015 Pro Phe Pro Gln Arg Pro Gln Gln Pro Phe Pro Gln Pro Gln 20 25 30

What is claimed is:
 1. A method of treating Celiac Sprue and/ordermatitis herpetiformis, the method comprising: administering to apatient an effective dose of a glutenase; wherein said glutenaseattenuates gluten toxicity in said patient.
 2. The method according toclaim 1, wherein said glutenase is an enzyme capable of cleaving apretreated substrate, wherein said pretreated substrate comprises one ormore gliadin, hordein, secalin or avenin proteins after pretreatmentwith physiological quantities of gastric and pancreatic proteases, andwherein said glutenase, when added to a reaction mixture comprising saidpretreated substrate, increases the concentration of free NH₂-termini byat least about 25%, and/or reduces the residual molar concentration ofoligopeptides greater than about 1000 Da by at least about 2-fold,and/or reduces the potency by which said pretreated substrateantagonizes binding of (SEQ ID NO:17) PQPELPYPQPQLP to HLA-DQ2.
 3. Themethod according to claim 1, wherein said glutenase is an enzyme havingat least about 20% sequence identity at the amino acid level to one of:prolyl endopeptidase from F. meningosepticum, DCP I from E. coli and DPPIV from Aspergillus fumigatus (Genbank accession number U87950).
 4. Themethod according to claim 1, wherein said glutenase has a specificactivity of at least 2.5 U/mg for cleavage of a peptide comprising oneof more motifs selected from the group consisting of Gly-Pro-pNA,Z-Gly-Pro-pNA, and Hip-His-Leu and/or a kcat/Km of at least about 2.5s⁻¹ M⁻¹ for cleavage of a peptide selected from the group consisting of(SEQ ID NO:1) QLQPFPQPQLPY, (SEQ ID NO:3) PQPQLPYPQPQLPY, (SEQ ID NO:13)QPQQSFPQQQ, (SEQ ID NO:14) QLQPFPQPELPY, (SEQ ID NO:15) PQPELPYPQPELPY,and (SEQ ID NO:16) QPQQSFPEQQ.
 5. The method according to claim 1,wherein said glutenase is an enzyme belonging to the classificationgroup EC 3.4.21.26, EC 3.4.14.5, or EC 3.4.15.1.
 6. The method accordingto claim 1, wherein said glutenase is formulated with a pharmaceuticallyacceptable excipient.
 7. The method according to claim 1, wherein saidglutenase is admixed with food.
 8. The method according to claim 1,wherein said glutenase is stable to acid conditions.
 9. The methodaccording to claim 1, wherein said glutenase is administered orally. 10.The method according to claim 9, wherein said glutenase is contained ina formulation that comprises an enteric coating.
 11. A formulation foruse in treatment of Celiac Sprue and/or dermatitis herpetiformis,comprising: an effective dose of glutenase and a pharmaceuticallyacceptable excipient.
 12. The formulation according to claim 11, whereinsaid glutenase is an enzyme capable of cleaving a pretreated substrate,wherein said pretreated substrate comprises one or more gliadin,hordein, secalin or avenin proteins after pretreatment withphysiological quantities of gastric and pancreatic proteases, whereinsaid glutenase, when added to a reaction mixture comprising saidpretreated substrate, increases the concentration of free NH₂-termini byat least about 25%, and/or reduces the residual molar concentration ofoligopeptides greater than about 1000 Da by at least about 2-fold,and/or reduces the potency by which said pretreated substrateantagonizes binding of (SEQ ID NO:17) PQPELPYPQPQLP to HLA-DQ2.
 13. Theformulation according to claim 12, wherein said glutenase is an enzymehaving at least about 20% sequence identity at the amino acid level toone of: prolyl endopeptidase from F. meningosepticum, DCP I from rabbitand DPP IV from Aspergillus fumigatus (Genbank accession number U87950).14. The formulation according to claim 12, wherein said glutenase has aspecific activity of at least 2.5 U/mg for cleavage of a peptidecomprising one of more motifs selected from the group consisting ofGly-Pro-pNA, Z-Gly-Pro-pNA, and Hip-His-Leu and/or a kcat/Km of at leastabout 2.5 s⁻¹ M⁻¹ for cleavage of a peptide selected from the groupconsisting of (SEQ ID NO:1) QLQPFPQPQLPY, (SEQ ID NO:3) PQPQLPYPQPQLPY,(SEQ ID NO:13) QPQQSFPQQQ, (SEQ ID NO:14) QLQPFPQPELPY, (SEQ ID NO:15)PQPELPYPQPELPY, and (SEQ ID NO:16) QPQQSFPEQQ.
 15. The formulationaccording to claim 12, wherein said glutenase is an enzyme belonging tothe classification group EC 3.4.21.26, EC 3.4.14.5, or EC 3.4.15.1. 16.The formulation according to claim 12, wherein said glutenase is stableto acid conditions.
 17. The formulation according to claim 12, whereinsaid formulation is suitable for oral administration.
 18. Theformulation according to claim 12, wherein said formulation comprises anenteric coating.
 19. A method for treating a foodstuff to render saidfoodstuff less toxic to a Celiac Sprue patient, said method comprisingcontacting said foodstuff with a glutenase.
 20. The method of claim 19,wherein said glutenase is prolyl endopeptidase.
 21. A purifiedoligopeptide selected from the group consisting of (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, a deamidated counterpartoligonucleotide, an analog oligopeptide, and a conjugate of tissuetransgluatonimase and a counterpart oligonucleotide.
 22. A method ofdiagnosing Celiac Sprue in an individual said method comprisingdetermining the presence of T cells in said individual that are reactivetowards to the oligopeptide of claim
 21. 23. A method of diagnosingCeliac Sprue in an individual said method comprising determining thepresence in said individual of antibodies specific for the oligopeptideof claim 21, or a derivative or conjugate thereof.
 24. A method fordiagnosing Celiac Sprue that comprises detecting the presence of anoligopeptide of claim 21 in a tissue, bodily fluid, or stool of anindividual.